CN106661121A

CN106661121A - Modified antigen binding polypeptide constructs and uses thereof

Info

Publication number: CN106661121A
Application number: CN201580036617.1A
Authority: CN
Inventors: M·桑彻斯; T·斯普雷特冯克罗登斯泰恩; D·厄洛塞夫; S·A·L·汤姆-耶; A·L·科珀; I·E·P·德安杰罗; Y-C·周; S·B·迪克西特
Original assignee: Zymeworks Inc Canada
Current assignee: Zymeworks BC Inc
Priority date: 2015-04-28
Filing date: 2015-05-29
Publication date: 2017-05-10
Also published as: JP2023027107A; JP7183221B2; JP7103751B2; KR20230019503A; JP7103751B6; JP2020202848A; JP2018502550A

Abstract

The present invention provides heterodimer pairs that can comprise a first heterodimer and a second heterodimer wherein each heterodimer comprises an immunoglobulin heavy chain or fragment thereof and an immunoglobulin light chain or fragment thereof. At least one of the heterodimers can comprise one or more amino acid modifications in the CH1 and/or CL domains, one or more amino acid modifications in the VH and/or VL domains, or a combination thereof. The modified amino acid(s) can be part of the interface between the light chain and heavy chain and are typically modified to create preferential pairing between each heavy chain and a desired light chain such that when the two heavy chains and two light chains of the heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains rather than the other. Likewise, the heavy chain of the second heterodimer typically preferentially pairs with the second light chain rather than first.

Description

Modified antigen-binding polypeptides construct and application thereof

The cross reference of related application

Patent application claims are filed in the U.S. Provisional Application No.62/003,663 on May 28th, 2014 and are filed in The U.S. Provisional Application No.62/154 on April 28th, 2015,055 priority, these patent applications are overall by reference It is expressly incorporated herein for all purposes.

Present patent application is related to be filed in the PCT/CA2013/050914 on November 28th, 2013, is filed in 2012 11 The U.S. Provisional Application No.61/730,906 on the moon 28, the U.S. Provisional Application No.61/761 for being filed on 2 6th, 2013, 641st, it is filed in the U.S. Provisional Application No.61/818,874 on May 2nd, 2013 and is filed in August, 2013 U.S. of 23 days Provisional application No.61/869,200, the complete disclosure of each of these patent applications is whole by reference accordingly Body is incorporated to for all purposes.

Sequence table

Sequence table of the present patent application comprising the electronic version submission with ASCII fromat, and accordingly by reference It is integrally incorporated.The ASCII copy creatings on May 29th, 2015, be named as 97993-945204 (000110PC) _ SL.txt, size is 27,012 byte.

Background

Bispecific antibody can be bound to two different epi-positions.The epi-position can be on identical antigen, or each table Position can be on different antigen.This feature of bispecific antibody becomes the noticeable work for various treatment uses Tool, wherein the antibody have the treatment beneficial effect more than a molecule is targetted or raised in disease treatment.Form double special Property antibody a kind of method by be related to two uniquenesses heavy chain of antibody and two uniquenesses light chain of antibody while express.Correct shape Challenge is remained into the bispecific antibody form similar to wild type, because heavy chain of antibody is related to be tied in the way of relative mixing Close light chain of antibody.Mix the result of pairing as this, expression while two heavy chain of antibody and two light chain of antibody is led naturally Heavy chain-light chain pairing is caused to mix.The mispairing remains the significant challenge for preparing bispecific therapeutic agent, wherein homogeneous pairing is good The basic demand of good technique and biological efficacy.

Various bispecific antibody preparation methods are described, wherein specific light chain of antibody or fragment are anti-with specific Body weight chain or fragment are matched.The commentary for solving the various methods of the problem is found in Klein etc., (2012) mAbs 4：6,1-11. International patent application No.PCT/EP2011/056388 (WO 2011/131746) is described for preparing heterodimeric body protein In-vitro method, wherein asymmetric mutation is introduced into the CH3 areas of two monospecifics starting albumen, to incubate under the reducing conditions Orientation " Fab arms " or " half molecule " between two monospecific IgG4 or IgG4 sample antibodies is driven to exchange when educating.

Schaefer etc. (Roche Diagnostics GmbH) is described in the case where manual splice is not used in the future Two heavy chains and two light chains for coming from two kinds of existing antibody are assembled into the method (PNAS of people's bivalent, bispecific IgG antibody (2011)108(27)：11187-11192).The method is related to the Fab of the half for exchanging bispecific antibody (Fab) heavy chain and light chain domain in.

Strop etc. (Rinat-Pfizer Inc.) is described by single expression and is purified two kinds of antibody of interest, so Afterwards they are mixed together into the method for preparing stable bispecific antibody under specified Redox Condition (J.Mol.Biol.(2012)420：204-19).

The engineered double antibody construct of Zhu etc. (Genentech) is (by the varistructure complete lack of constant domain Domain antibodies fragment is constituted) VL/VH interfaces in mutation, and be prepared for heterodimer double antibody (Protein Science (1997)6：781-788).Similarly, in the also engineered VL/VH interfaces of single-chain diabodies of Igawa etc. (Chugai) Be mutated with contribute to double antibody selective expression and suppress double antibody stereo isomers (Protein Engineering, Design&Selection(2010)23：667-677).

United States Patent (USP) discloses No.2009/0182127 (Novo Nordisk, Inc.) and describes by modifying light-heavy chain To Fc interfaces and CH1：Preparing bispecific antibody, the modification reduces a pair of light chain to the amino acid residue of CL interfaces The ability that heavy chain with another pair interacts.

United States Patent (USP) discloses No.2014/0370020 (Chugai) and describes by replacing CH1 and CL with Charged acids Amino acid present on interface between area is adjusting the association between these areas of antibody.

General introduction

This document describes the detached antigen-binding polypeptides comprising at least the first heterodimer and the second heterodimer Construct, first heterodimer includes the first heavy chain immunoglobulin peptide sequence (H1) and the first light chain immunoglobulin Peptide sequence (L1)；And the second heterodimer includes the second heavy chain immunoglobulin peptide sequence (H2) and the second immune ball Protein light chain peptide sequence (L2), wherein at least one of H1 or L1 sequences of the first heterodimer are heterologous different from second Dimeric correspondence H2 or L2 sequences, and each self-contained at least heavy-chain variable domains (V of wherein H1 and H2_HDomain) and weight Chain constant domain (C_H1Domain)；The each self-contained at least light variable domains (V of L1 and L2_LDomain) and chain constant knot Structure domain (C_L)；And at least one of H1, H2, L1 and L2 are variable comprising at least one constant domain and/or at least one At least one of domain is amino acid modified, wherein compared with L2 H1 preferentially with L1 match, and compared with L1 H2 preferentially with L2 is matched.

In some respects, the construct also includes heterodimer Fc, and the Fc includes at least two C_H3Sequence, the wherein Fc By or by one or more joints be not coupled to the first heterodimer and the second heterodimer, wherein dimerization C_H3Sequence Row have about 68 DEG C or higher of melt temperature (Tm) (as differential scanning calorimetry (DSC) is determined), and the wherein structure Body is bispecific.

In some respects, at least one amino acid modified at least one amino acid selected from shown in form or embodiment is repaiied Decorations.

In some respects, when H1, H2, L1 and L2 are co-expressed in cell or mammalian cell, or as H1, H2, L1 When being co-expressed in Cell free expression system with L2, or when H1, H2, L1 and L2 symbiosis into when, or when H1, H2, L1 and L2 via The symbiosis of redox generation method into when, compared with L2 H1 preferentially with L1 match, and compared with L1 H2 preferentially with L2 match.

In some respects, at least one of H1, H2, L1 and L2 include V_HAnd/or V_LAt least one amino acid of domain Modification and C_H1And/or C_LAt least one of domain is amino acid modified so that H1 is preferentially matched with L1 compared with L2, and/or H2 is preferentially matched with L2 compared with L1.

In some respects, if H1 includes C_H1At least one of domain is amino acid modified, then in L1 and L2 at least One includes C_LAt least one of domain is amino acid modified；And/or if H1 includes V_HAt least one of domain amino Acid modification, then at least one of L1 and L2 include V_LAt least one of domain is amino acid modified.

In some respects, H1, L1, H2 and/or L2 include at least 1,2,3,4,5,6,7,8,9 or 10 amino acid mutations. In some respects, at least one of H1, H2, L1 and L2 include at least one constant domain and/or at least one variable knot At least 2,3,4,5,6,7,8,9 or 10 of structure domain are amino acid modified.

In some respects, when both L1 and L2 are co-expressed with least one of H1 and H2, H1-L1 and H2-L2 is heterologous Dimer at least one of opposing pairs and each self-corresponding H1-L2 or H2-L1 heterodimers to ratio be more than 50%th, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%th, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%th, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%th, 96%, 97%, 98% or 99%, and the opposing pairs of wherein modified H1-L1 or H2-L2 heterodimers pair Each match somebody with somebody relatively more than what is observed without at least one amino acid modified corresponding H1-L1 or H2-L2 heterodimers centering It is right.

In some respects, the heat with melt temperature (Tm) metering of at least one of first and second heterodimers is steady Qualitative (as DSF is determined) without at least one amino acid modified corresponding heterodimer Tm about 0,1,2,3,4,5, 6th, within 7,8,9 or 10 DEG C.In some respects, each comprising at least one amino acid modified heterodimer melting temperature The heat endurance (as DSF is determined) of degree (Tm) metering is in the Tm without at least one amino acid modified corresponding heterodimer About 0,1,2,3,4,5,6,7,8,9 or 10 DEG C within.In some embodiments, each is amino acid modified comprising at least one Heterodimer the heat endurance (as DSF is determined) measured with melt temperature (Tm) it is at least one amino acid modified in nothing About 0,1,2 or 3 DEG C of Tm of corresponding heterodimer within.

In some respects, the affinity of the antigen that each heterodimer is combined to it is each not modified heterologous two Aggressiveness in about 1,2,3,4,5,6,7,8,9,10 times of the affinity of identical antigen (such as surface plasmon resonance (SPR) Or FACS is determined).

In some respects, at least one of H1 and L1 include domain as at least one：It includes at least one It is amino acid modified, produce bigger amino acid spatial complementary as H1 and L1 non-matchings compared with L2.In some respects, H2 and L2 At least one include domain as at least one：Its include it is at least one amino acid modified, as H2 and L2 compared with L1 Bigger amino acid spatial complementary is produced during pairing.In some respects, at least one of H1 and L1 include at least one so Domain：It includes at least one amino acid modified, and bigger Charged acids are produced when H1 and L1 is matched compared with L2 Between electrostatic complementarities.In some respects, at least one of H2 and L2 include domain as at least one：Its include to Few one amino acid modified, the electrostatic complementarities compared with L1 when H2 and L2 is matched between the bigger Charged acids of generation.

In some respects, at least one amino acid modified be one group shown at least one of form or embodiment and dash forward Become.

In some respects, the construct also includes Fc, and the Fc includes at least two C_H3Sequence, the wherein Fc pass through or obstructed Cross one or more joints and be coupled to the first heterodimer and the second heterodimer.

In some respects, the Fc be people Fc, human IgG1 Fc, people IgA Fc, human IgG Fc, people IgD Fc, people IgE Fc, People IgM Fc, human IgG2 Fc, human IgG 3Fc or human IgG 4Fc.In some respects, the Fc is heterodimer Fc.In some sides Face, the Fc includes C_H3One or more modifications at least one of sequence.In some respects, dimerization C_H3Sequence has There are about 68,69,70,71,72,73,74,75,76,77,77.5,78,79,80,81,82,83,84 or 85 DEG C or higher melting Temperature (Tm) (as DSC is determined).In some respects, in the preparation, the Fc be to be greater than about 75%, 76%, 77%, 78%, 79%th, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%th, the heterodimer that 95%, 96%, 97%, 98% or 99% purity is formed；Or wherein in expression or by single When individual cell is expressed, the Fc be to be greater than about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%th, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% The heterodimer that purity is formed.In some respects, the Fc includes C_H3One or more modifications at least one of sequence, The modification contributes to forming heterodimer Fc of the stability equivalent to wild-type homologous dimeric Fc.In some respects, the Fc Also include at least one C_H2Sequence.In some respects, the C of the Fc_H2Sequence includes one or more modifications.In some respects, should One or more modifications of the Fc comprising the selective binding for contributing to Fc- γ acceptors.

In some embodiments, the Fc is included：

I) there is the modification T366L_ in the modification L351Y_F405A_Y407V and the 2nd Fc polypeptides in a Fc polypeptides The heterodimer IgG1 Fc of K392M_T394W；

Ii) there is the modification T366L_ in the modification L351Y_F405A_Y407V and the 2nd Fc polypeptides in a Fc polypeptides The heterodimer IgG1Fc of K392L_T394W；

Iii) there is the modification in the modification T350V_L351Y_F405A_Y407V and the 2nd Fc polypeptides in a Fc polypeptides The heterodimer IgG1Fc of T350V_T366L_K392L_T394W；

Iv) there is the modification in the modification T350V_L351Y_F405A_Y407V and the 2nd Fc polypeptides in a Fc polypeptides The heterodimer IgG1Fc of T350V_T366L_K392M_T394W；Or

V) during there is the modification T350V_L351Y_S400E_F405A_Y407V and the 2nd Fc polypeptides in a Fc polypeptides The heterodimer IgG1Fc of modification T350V_T366L_N390R_K392M_T394W.

In some respects, the Fc is coupled to heterodimer by one or more joints, or the wherein Fc passes through one Or multiple joints are coupled to H1 and H2.In some respects, one or more of joints are one or more peptide linkers.One A little aspects, one or more of joints include one or more antibody hinge regions.In some respects, it is one or more of to connect Head includes one or more IgG1 hinge areas.In some respects, one or more of joints include one or more modifications. Some aspects, the one or more modifications of one or more of joints contribute to the selective binding of Fc- γ acceptors.

In some respects, at least one it is amino acid modified be at least one amino acid mutation, or wherein at least one amino Acid modification is at least one amino acid replacement.

In some respects, the sequence of each in H1, H2, L1 and L2 derives from human sequence.

In some respects, construct is polyspecific or bispecific.In some respects, construct be multivalence or Divalence.

In some respects, heterodimer as herein described preferentially matches to form bispecific antibody.For example, at some In embodiment, heavy chain polypeptide sequence H1 and H2 include total length heavy chain sequence, and the total length heavy chain sequence includes light chain constant structure Domain (C_H1Domain), C_H2Domain and C_H3Domain.In some embodiments, bispecific antibody (for example, H1-L1：H2- L2 in) the correct heavy chain of pairing and the percentage of light chain more than 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%th, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%th, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%th, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

There is also described herein the separation comprising at least one sequence for encoding construct as herein described or heavy chain or light chain Polynucleotides or detached polynucleotides group.In some respects, the polynucleotides or polynucleotides group are cDNA.Also retouch herein State comprising the carrier of one or more or vehicle group in polynucleotides as herein described or polynucleotides group.In some sides Face, the carrier or vehicle group are selected from plasmid, polycistronic vector, viral vector, non-free type mammalian vector, expression vector And recombinant expression carrier.

There is also described herein comprising polynucleotides as herein described or polynucleotides group or carrier as herein described or carrier The detached cell of group.In some respects, the cell is hybridoma, Chinese hamster ovary (CHO) cell or HEK293 cells.

There is also described herein the pharmaceutical composition comprising construct as herein described and pharmaceutically acceptable carrier.One A little aspects, said composition also comprising one or more selected from buffer solution, antioxidant, low-molecular-weight molecule, medicine, protein, The material of amino acid, carbohydrate, lipid, chelating agent, stabilizer and excipient.

There is also described herein construct as herein described or pharmaceutical composition as herein described are used to treat in experimenter Disease or illness or cancer or vascular diseases or for the purposes in medicine manufacture.

There is also described herein the method for experimenter of the treatment with disease or illness or cancer or vascular diseases, it include to Experimenter applies construct as herein described or composition as herein described.

There is also described herein the method for obtaining construct as herein described from host cell cultures, the method includes following Step：A () obtains the host cell cultures comprising at least one host cell, the host cell includes one or more coding The nucleotide sequence of the construct；And (b) reclaims the construct from the host cell cultures.

There is also described herein the method for obtaining construct as herein described, it is comprised the following steps：A () obtains H1, L1, H2 Or L2；B () allows the H1 compared with L2 preferentially to match with L1, and H2 is preferentially matched with L2 compared with L1；And (c) is somebody's turn to do Construct.

There is also described herein the method for preparing construct as herein described, it includes：Obtain at least one construct of coding Polynucleotides or polynucleotides group；It is determined that in for being introduced into the polynucleotides of at least one host cell or polynucleotides group The best ratio of each, the wherein best ratio are by assessment and the H1- of the mispairing formed when H1, L1, H2 or L2 are expressed L2 with H2-L1 heterodimers are to comparing H1-L1 the and H2-L2 heterodimers formed when H1, L1, H2 or L2 are expressed to phase For amount determining；Preferred best ratio is selected, wherein with the polynucleotides or many of the preferred best ratio The transfection at least one host cell of nucleotides group causes the construct to be expressed；With the polynucleotides or many nucleosides of best ratio At least one host cell of acid group transfection；And at least one host cell is cultivated to express the construct.

In some respects, the selection of best ratio is assessed by the transfection in transient transfection system.In some respects, use The polynucleotides or at least one host cell of polynucleotides group transfection of preferred best ratio cause construct optimum expression. Some aspects, the construct includes Fc, and the Fc includes at least two C_H3Sequence, the wherein Fc pass through or do not pass through one or more Joint is coupled to the first heterodimer and the second heterodimer.In some respects, the Fc is heterodimer, is optionally wrapped It is amino acid modified containing one or more.

There is also described herein the computer-readable recording medium of data storage collection, the data set is comprising representing first heterologous two The data of the complemented mutant in aggressiveness and the second heterodimer, first heterodimer includes the first heavy chain immunoglobulin Peptide sequence (H1) and the first light chain immunoglobulin peptide sequence (L1)；And second heterodimer is comprising the second immune ball Ferritin heavy chain peptide sequence (H2) and the second light chain immunoglobulin peptide sequence (L2), wherein H1 and H2 is each self-contained at least to be weighed Chain variable domains (V_HDomain) and heavy chain constant domain (C_H1Domain)；The each self-contained at least light chains of wherein L1 and L2 can Structure changes domain (V_LDomain) and light chain constant domain (C_LDomain), and the wherein data set of complemented mutant is comprising expression Those mutation listed in form or embodiment or the data of the subset of those mutation；And for determining that H1 is preferential compared with L2 The computer-executable code of ground possibility that H2 is preferentially matched with L2 with L1 pairings and/or compared with L1.

There is also described herein the computer-implemented method for determining preferential pairing, it includes：Obtain data set, the number According to collection comprising the data for representing the first heterodimer and the complemented mutant in the second heterodimer, first heterodimer Comprising the first heavy chain immunoglobulin peptide sequence (H1) and the first light chain immunoglobulin peptide sequence (L1)；And this is second different Source dimer includes the second heavy chain immunoglobulin peptide sequence (H2) and the second light chain immunoglobulin peptide sequence (L2), its The each self-contained at least heavy-chain variable domains (V of middle H1 and H2_HDomain) and heavy chain constant domain (C_H1Domain)；Wherein L1 Self-contained at least light variable domains (V each with L2_LDomain) and light chain constant domain (C_LDomain), and it is wherein mutual Mend the data of the data set comprising the subset for representing those mutation or those mutation listed in form or embodiment of mutation；And By computer processor determine compared with L2 H1 preferentially with L1 pairing and/or compared with L1 H2 preferentially with L2 pairing can Can property.In some respects, the method also includes preparing construct as herein described.

There is also described herein the method for preparing bispecific antigen-binding polypeptides construct, the bispecific construct bag Containing the first heterodimer and the second heterodimer, first heterodimer is included from the first monospecific antigen binding The first heavy chain immunoglobulin peptide sequence (H1) and the first light chain immunoglobulin peptide sequence (L1) of polypeptide；And second is different Source dimer includes the second heavy chain immunoglobulin peptide sequence (H2) from the second monospecific antigen-binding polypeptides and the Two light chain immunoglobulin peptide sequences (L2), each self-contained at least heavy-chain variable domains (V of wherein H1 and H2_HDomain) and Heavy chain constant domain (C_H1Domain)；The each self-contained at least light variable domains (V of wherein L1 and L2_LDomain) and light chain Constant domain (C_LDomain), the method includes：To draw from one or more complemented mutants of data set as herein described Enter the first heterodimer and/or the second heterodimer；And the first heterodimer and the second heterodimer are made extremely It is co-expressed in a kind of few host cell, to prepare the expression product comprising bispecific construct.

In some respects, the method also includes determining that the expression product builds relative to bispecific in other polypeptide products The amount of body, to select preferred complemented mutant subset.In some respects, the bispecific construct is produced compared to other polypeptides Thing, with more than 70% (for example, more than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%th, 98% or purity 99%) generate.In some respects, the data set is data set as herein described.In some respects, The method is also included at least one of other amino acid modified addition to H1, H2, L1 or L2, to improve bispecific structure Build body phase compared with other polypeptide products purity the step of.In some respects, the construct includes Fc, and the Fc includes at least two C_H3Sequence, the wherein Fc by or by one or more joints be not coupled to the first heterodimer and the second heterodimeric Body.In some respects, the Fc is heterodimer, optionally amino acid modified comprising one or more.In some respects, this resists Former Binding peptide is antibody, Fab or scFv.

In some embodiments of the construct, H1 and/or H2 is comprising at L124, K145, D146, Q179 and S186 At least one or one group amino acid modified, and L1 and/or L2 includes Q124, S131, V133, Q160, S176, T178 and T180 At least one or one group of place is amino acid modified.For example, in some embodiments, H1 and/or H2 comprising selected from L124R, At least one or one group of L124E, K145M, K145T, D146N, Q179E, Q179K, S186R and S186K is amino acid modified, and And L1 and/or L2 comprising selected from Q124E, S131R, S131K, V133G, Q160E, S176R, S176D, T178D, T178E and At least one or one group of T180E is amino acid modified.In some embodiments, H1 is comprising selected from L124E, K145M, K145T It is amino acid modified with Q179E or combinations thereof；L1 is comprising selected from S131R, S131K, V133G and S176R or their group That what is closed is amino acid modified；H2 is repaiied comprising the amino acid selected from L124R, D146N, Q179K, S186R and S186K or combinations thereof Decorations；And L2 includes the amino selected from Q124E, V133G, Q160E, S176D, T178D, T178E and T180E or combinations thereof Acid modification.In some embodiments, H1 includes amino acid modified L124E, K145T and Q179E；L1 is comprising amino acid modified S131K, V133G and S176R；H2 includes amino acid modified L124R and S186R；And L2 comprising amino acid modified V133G, S176D and T178D.

In some embodiments of the construct, H1 and/or H2 comprising L124, L143, K145, D146, Q179 and At least one or one group at S186 is amino acid modified, and L1 and/or L2 comprising Q124, V133, Q160, S176, T178 and At least one or one group at T180 is amino acid modified.In some embodiments, H1 and/or H2 comprising selected from L124E, At least one or one group of amino acid of L124R, L143E, L143D, K145T, K145M, D146N, Q179K, S186R and S186K Modification；And L1 and/or L2 comprising selected from Q124K, Q124E, V133G, Q160K, S176R, S176D, T178E, T178K, At least one or one group of T178R, T178D and T180E is amino acid modified.In some embodiments, H1 is included and is selected from L124E, L143E, L143D, K145T and K145M or combinations thereof it is amino acid modified；L1 comprising selected from Q124K, V133G, Q160K, S176R, T178K and T178R or combinations thereof it is amino acid modified；H2 comprising selected from L124R, D146N, Q179K, S186R and S186K or combinations thereof it is amino acid modified；And L2 comprising selected from Q124E, V133G, S176D, T178E, T178D and T180E or combinations thereof it is amino acid modified.In some embodiments, H1 comprising amino acid modified L124E, L143E and K145T；L1 includes amino acid modified Q124K, V133G and S176R；H2 includes amino acid modified L124R and Q179K； And L2 includes amino acid modified V133G, S176D and T178E.In some embodiments, H1 is comprising amino acid modified L124E, L143E and K145T；L1 includes amino acid modified Q124K, V133G and S176R；H2 comprising amino acid modified L124R and S186R；And L2 includes amino acid modified V133G, S176D and T178D.

In some embodiments of the construct, H1 and/or H2 includes Q39, L45, L124, L143, F122 and H172 Place at least one or one group it is amino acid modified, and L1 and/or L2 comprising Q38, P44, Q124, S131, V133, N137, At least one or one group at S174, S176 and T178 is amino acid modified.In some embodiments, H1 and/or H2 includes choosing Repair from least one or one group of amino acid of Q39E, Q39R, L45P, F122C, L124E, L124R, L143F, H172T and H172R Decorations；And L1 and/or L2 comprising selected from Q38R, Q38E, P44F, Q124C, S131T, S131E, V133G, N137K, S174R, At least one or one group of S176R, S176K, S176D, T178Y and T178D is amino acid modified.In some embodiments, H1 Comprising selected from the amino acid modified of Q39E, L45P, F122C, L124E, L143F, H172T and H172R or combinations thereof；L1 bags Containing selected from Q38R, P44F, Q124C, S131T, V133G, N137K, S174R, S176R, S176K and T178Y or combinations thereof It is amino acid modified；H2 is comprising selected from the amino acid modified of Q39R, L124R and H172R or combinations thereof；And L2 includes choosing From the amino acid modified of Q38E, S131E, V133G, S176D and T178D or combinations thereof.In some embodiments, H1 bags Containing amino acid modified Q39E and L124E；L1 includes amino acid modified Q38R, V133G and S176R；H2 is comprising amino acid modified Q39R and L124R；And L2 includes amino acid modified Q38E, V133G and S176D.In some embodiments, H1 includes amino Acid modification L45P and L124E；L1 includes amino acid modified P44F, V133G and S176R；H2 includes amino acid modified L124R；And And L2 includes amino acid modified V133G, S176D and T178D.In some embodiments, H1 comprising amino acid modified L124E and L143F；L1 includes amino acid modified V133G and S176R；H2 includes amino acid modified L124R；And L2 is comprising amino acid modified V133G, S176D and T178D.In some embodiments, H1 includes amino acid modified F122C and L124E；L1 includes amino acid Modification Q124C, V133G and S176R；H2 includes amino acid modified L124R；And L2 comprising amino acid modified V133G and S176D.In some embodiments, H1 includes amino acid modified L124E and H172T；L1 comprising amino acid modified V133G, N137K, S174R and S176R；H2 includes amino acid modified L124R and H172R；And L2 comprising amino acid modified V133G, S176D and T178D.

In some embodiments of the construct, H1 and/or H2 is included at L124, A125, H172 and K228 at least One or one group amino acid modified, and L1 and/or L2 is included at S121, V133, N137, S174, S176 and T178 at least One or one group amino acid modified.In some embodiments, H1 and/or H2 comprising selected from L124E, L124R, A125S, At least one or one group of A125R, H172R, H172T and K228D is amino acid modified；And (ii) L1 and/or L2 is included and is selected from At least one or one group of S121K, V133G, N137K, S174R, S176K, S176R, S176D and T178D is amino acid modified. In some embodiments, H1 is comprising selected from the amino acid modified of L124E, A125S, H172R and K228D or combinations thereof；L1 Comprising selected from the amino acid modified of S121K, V133G and S176R or combinations thereof；H2 comprising selected from L124R, A125R and H172T or combinations thereof it is amino acid modified；And L2 comprising selected from V133G, N137K, S174R, S176D and T178D or Combinations thereof it is amino acid modified.In some embodiments, H1 includes amino acid modified L124E and K228D；L1 includes ammonia Base acid modification S121K, V133G and S176R；H2 includes amino acid modified L124R and A125R；And L2 is comprising amino acid modified V133G and S176D.In some embodiments, H1 includes amino acid modified L124E and H172R；L1 is comprising amino acid modified V133G and S176R；H2 includes amino acid modified L124R and H172T；And L2 comprising amino acid modified V133G, S174R and S176D。

In some embodiments of the construct, H1 and/or H2 comprising at least one at L124, A139 and V190 or One group amino acid modified, and L1 and/or L2 is comprising at least one or one group of amino acid at F116, V133, L135 and S176 Modification.In some embodiments, H1 and/or H2 includes at least selected from L124E, L124R, A139W, A139G and V190A It is individual or one group it is amino acid modified；And L1 and/or L2 is comprising selected from F116A, V133G, L135V, L135W, S176R and S176D At least one or one group it is amino acid modified.In some embodiments, H1 is comprising selected from L124E and A139W or their group That what is closed is amino acid modified；L1 is comprising selected from the amino acid modified of F116A, V133G, L135V and S176R or combinations thereof；H2 Comprising selected from the amino acid modified of L124R, A139G and V190A or combinations thereof；And L2 is comprising selected from V133G, L135W It is amino acid modified with S176D or combinations thereof.In some embodiments, H1 comprising amino acid modified L124E and A139W；L1 includes amino acid modified F116A, V133G, L135V and S176R；H2 comprising amino acid modified L124R, A139G and V190A；And L2 includes amino acid modified V133G, L135W and S176D.

In some embodiments of the construct, H1 and/or H2 includes Q39, L45, K145, H172, Q179 and S186 At least one or one group of place is amino acid modified, and L1 and/or L2 comprising Q38, P44, Q124, S131, Q160, T180 and At least one or one group at C214 is amino acid modified.In some embodiments, H1 and/or H2 comprising selected from Q39E, Q39R, At least one or one group of L45P, K145T, H172R, Q179E and S186R is amino acid modified；And L1 and/or L2 is included and is selected from At least one or one group of Q38R, Q38E, P44F, Q124E, S131K, Q160E, T180E and C214S is amino acid modified.One In a little embodiments, H1 is comprising selected from the amino acid modified of Q39E, L45P, K145T, H172R and Q179E or combinations thereof； L1 is comprising selected from the amino acid modified of Q38R, P44F and S131K or combinations thereof；H2 comprising selected from Q39R, H172R and S186R or combinations thereof it is amino acid modified；And L2 comprising selected from Q38E, Q124E, Q160E, T180E and C214S or it Combination it is amino acid modified.In some embodiments, H1 includes amino acid modified Q39E, K145T and Q179E；L1 bags Containing amino acid modified Q38R and S131K；H2 includes amino acid modified Q39R and S186R；And L2 comprising amino acid modified Q38E, Q124E, Q160E and T180E.In some embodiments, H1 includes amino acid modified L45P, K145T, H172R and Q179E； L1 includes amino acid modified P44F and S131K；H2 includes amino acid modified H172R and S186R；And L2 is comprising amino acid modified Q124E, Q160E and T180E.

In some embodiments of the construct, H1 and/or H2 is comprising at A139, L143, K145, Q179 and V190 At least one or one group amino acid modified, and L1 and/or L2 is comprising at F116, Q124, L135, Q160, T178 and T180 At least one or one group amino acid modified.In some embodiments, H1 and/or H2 comprising selected from A139W, A139G, L143E, At least one or one group of K145T, Q179E, Q179K and V190A is amino acid modified；And L1 and/or L2 is included and is selected from At least one or one group of F116A, Q124R, Q124E, L135V, L135W, Q160E, T178R and T180E is amino acid modified. In some embodiments, H1 is comprising selected from the amino acid modified of A139W, L143E, K145T and Q179E or combinations thereof；L1 Comprising selected from the amino acid modified of F116A, Q124R, L135V and T178R or combinations thereof；H2 comprising selected from A139G, Q179K and V190A or combinations thereof it is amino acid modified；And L2 comprising selected from Q124E, L135W, Q160E and T180E or Combinations thereof it is amino acid modified.In some embodiments, H1 comprising amino acid modified A139W, L143E, K145T and Q179E；L1 includes amino acid modified F116A, Q124R, L135V and T178R；H2 includes amino acid modified Q179K；And L2 bags Containing amino acid modified Q124E, L135W, Q160E and T180E.

In some embodiments of the construct, H1 and/or H2 includes Q39, L143, K145, D146, H172 and Q179 At least one or one group of place is amino acid modified, and L1 and/or L2 is included at Q38, Q124, Q160, T178 and T180 extremely Few one or one group amino acid modified.In some embodiments, H1 and/or H2 comprising selected from Q39E, Q39R, L143E, At least one or one group of K145T, D146G, H172R, Q179E and Q179K is amino acid modified；And L1 and/or L2 includes choosing At least one or one group from Q38R, Q38E, Q124R, Q124E, Q160K, Q160E, T178R and T180E is amino acid modified. In some embodiments, H1 is repaiied comprising the amino acid selected from Q39E, L143E, K145T, H172R and Q179E or combinations thereof Decorations；L1 is comprising selected from the amino acid modified of Q38R, Q124R, Q160K and T178R or combinations thereof；H2 comprising selected from Q39R, H172R and Q179K or combinations thereof it is amino acid modified；And L2 comprising selected from Q38E, Q124E, D146G, Q160E and T180E or combinations thereof it is amino acid modified.In some embodiments, H1 comprising amino acid modified Q39E, L143E, K145T and Q179E；L1 includes amino acid modified Q38R, Q124R, Q160K and T178R；H2 comprising amino acid modified Q39R, H172R and Q179K；And L2 includes amino acid modified Q38E, Q124E, Q160E and T180E.

In some embodiments of the construct, H1 and/or H2 includes L45, L143, K145, D146, H172 and Q179 Place at least one or one group it is amino acid modified, and L1 and/or L2 comprising Q38, P44, Q124, N137, Q160, S174, At least one or one group at T178, T180 and C214 is amino acid modified.In some embodiments, H1 and/or H2 includes choosing At least one or one group from L45P, L143E, K145T, D146G, H172R, H172T, Q179E and Q179K is amino acid modified； And (ii) L1 and/or L2 comprising selected from Q38E, P44F, Q124R, Q124E, N137K, Q160K, Q160E, S174R, T178R, At least one or one group of T180E and C214S is amino acid modified.In some embodiments, H1 comprising selected from L45P, L143E, K145T, H172R and Q179E or combinations thereof it is amino acid modified；L1 is comprising selected from P44F, Q124R, Q160K and T178R Or combinations thereof is amino acid modified；H2 includes the amino selected from D146G, H172R, H172T and Q179K or combinations thereof Acid modification；And L2 is comprising selected from Q38E, Q124E, N137K, Q160E, S174R, T180E and C214S or combinations thereof It is amino acid modified.In some embodiments, H1 includes amino acid modified L45P, L143E and K145T；L1 is repaiied comprising amino acid Decorations P44F, Q124R, Q160K and T178R；H2 includes amino acid modified D146G and Q179K；And L2 is comprising amino acid modified Q38E, Q124E, Q160E and T180E.In some embodiments, H1 includes amino acid modified L143E, K145T and H172R； L1 includes amino acid modified Q124R, Q160K and T178R；H2 includes amino acid modified H172T and Q179K；And L2 includes ammonia Base acid modification Q124E, Q160E, N137K, S174R and T180E.

In some embodiments of the construct, H1 and/or H2 is included at L124, L143, K145 and Q179 at least One or one group amino acid modified, and L1 and/or L2 is included at Q124, S131, V133, S176, T178 and T180 at least One or one group amino acid modified.In some embodiments, H1 and/or H2 comprising selected from L124W, L124A, L143E, At least one or one group of L143F, K145T, Q179E and Q179K is amino acid modified；And L1 and/or L2 is included and is selected from At least one of Q124R, Q124K, Q124E, S131K, V133A, V133W, S176T, T178R, T178L, T178E and T180E Or one group amino acid modified.In some embodiments, H1 comprising selected from L124W, L143E, K145T and Q179E or they That what is combined is amino acid modified；L1 comprising selected from Q124R, Q124K, S131K, V133A, S176T, T178R and T178L or they That what is combined is amino acid modified；H2 is comprising selected from the amino acid modified of L124A, L143F and Q179K or combinations thereof；And L2 Comprising selected from the amino acid modified of Q124E, V133W, S176T, T178L, T178E and T180E or combinations thereof.In some realities In applying scheme, H1 includes amino acid modified L124W, L143E, K145T and Q179E；L1 comprising amino acid modified Q124R, V133A, S176T and T178R；H2 includes amino acid modified L124A, L143F and Q179K；And L2 is comprising amino acid modified Q124E, V133W, S176T, T178L and T180E.

In some embodiments of the construct, H1 and/or H2 is comprising at A139, L143, K145, Q179 and S186 At least one or one group amino acid modified, and L1 and/or L2 is comprising at F116, Q124, V133, Q160, T178 and T180 At least one or one group amino acid modified.In some embodiments, H1 and/or H2 comprising selected from A139C, L143E, L143D, At least one or one group of amino acid of L143R, L143K, K145T, Q179E, Q179D, Q179R, Q179K, S186K, S186R is repaiied Decorations；And L1 and/or L2 comprising selected from F116C, Q124R, Q124K, Q124E, V133E, V133D, Q160K, Q160E, At least one or one group of T178R, T178K, T178E and T180E is amino acid modified.In some embodiments, H1 includes choosing From the amino acid modified of A139C, L143E, L143D, K145T, Q179E and Q179D or combinations thereof；L1 is included and is selected from F116C, Q124R, Q124K, Q160K, T178R and T178K or combinations thereof it is amino acid modified；H2 comprising selected from L143R, L143K, Q179R, Q179K, S186K and S186R or combinations thereof it is amino acid modified；And L2 comprising selected from Q124E, V133E, V133D, Q160E, T178E and T180E or combinations thereof it is amino acid modified.In some embodiments, H1 bags Containing amino acid modified A139C, L143E, K145T and Q179E；L1 includes amino acid modified F116C, Q124R and T178R；H2 bags Containing amino acid modified Q179K；And L2 includes amino acid modified Q124E, Q160E and T180E.In some embodiments, H1 Comprising amino acid modified L143E, K145T and Q179E；L1 includes amino acid modified Q124R and T178R；H2 is repaiied comprising amino acid Decorations S186K；And L2 includes amino acid modified Q124E, Q160E and T178E.In some embodiments, H1 includes amino acid Modification L143E, K145T and Q179E；L1 includes amino acid modified Q124R and T178R；H2 includes amino acid modified L143R；And And L2 includes amino acid modified Q124E and V133E.

In some embodiments of the construct, H1 and/or H2 includes L124, L143, K145, D146, Q179, S186 It is amino acid modified with least one or a group at S188, and L1 and/or L2 comprising Q124, S131, V133, Q160, S176, At least one or one group at T178 and T180 is amino acid modified.In some embodiments, H1 and/or H2 is included and is selected from L124A, L143A, L143R, L143E, L143K, K145T, D146G, Q179R, Q179E, Q179K, S186R, S186K and At least one or one group of S188L is amino acid modified；And L1 and/or L2 comprising selected from Q124R, Q124E, S131E, S131T, V133Y, V133W, V133E, V133D, Q160E, Q160K, Q160M, S176L, T178R, T178E, T178F, T178Y and At least one or one group of T180E is amino acid modified.In some embodiments, H1 is comprising selected from L143E, K145T, Q179E It is amino acid modified with S188L or combinations thereof；L1 includes the ammonia selected from Q124R, Q160K and T178R or combinations thereof Base acid modification；H2 comprising selected from L124A, L143A, L143R, L143K, D146G, Q179R, Q179K, S186R and S186K or it Combination it is amino acid modified；And L2 comprising selected from Q124E, S131E, S131T, V133Y, V133W, V133E, V133D, Q160E, Q160M, S176L, T178E, T178F, T178Y and T180E or combinations thereof it is amino acid modified.In some enforcements In scheme, H1 includes amino acid modified L143E, K145T, Q179E and S188L；L1 includes amino acid modified Q124R and T178R； H2 includes amino acid modified S186K；And L2 includes amino acid modified Q124E, S176L and T180E.In some embodiments In, H1 includes amino acid modified L143E, K145T, Q179E and S188L；L1 includes amino acid modified Q124R and T178R；H2 bags Containing amino acid modified S186K；And L2 includes amino acid modified Q124E, S131T, T178Y and T180E.In some embodiments In, H1 includes amino acid modified L143E and K145T；L1 includes amino acid modified Q124R, Q160K and T178R；H2 includes amino Acid modification S186K；And L2 includes amino acid modified S131E.In some embodiments, H1 includes amino acid modified L143E And K145T；L1 includes amino acid modified Q124R；H2 includes amino acid modified L143R；And L2 includes amino acid modified Q124E And V133E.

In some embodiments of the construct, H1 is repaiied comprising at least one or one group of amino acid at F122 and C233 Decorations, and L1 is amino acid modified comprising at least one or a group at Q124 and C214.In some embodiments, H1 includes choosing At least one or one group from F122C and C233S is amino acid modified；And L1 includes at least one selected from Q124C and C214S Or one group amino acid modified.In some embodiments, H1 includes the amino acid selected from F122C and C233S or combinations thereof Modification；L1 is comprising selected from the amino acid modified of Q124C and C214S or combinations thereof；H2 includes wild type or not modified Amino acid sequence；And L2 includes wild type or not modified amino acid sequence.In some embodiments, H1 includes amino Acid modification F122C and C233S；L1 includes amino acid modified Q124C and C214S；H2 includes wild type or not modified amino Acid sequence；And L2 includes wild type or not modified amino acid sequence.

In some embodiments, the construct comprising selected from this paper form SMCA design 9561-9095_1, 9561-9095_2、9121-9373_1、9121-9373_2、9116-9349_1、9116-9349_2、9134-9521_1、9134- 9521_2、9286-9402_1、9286-9402_2、9667-9830_1、9667-9830_2、9696-9848_1、9696-9848_ 2、9060-9756_1、9060-9756_2、9682-9740_1、9682-9740_2、9049-9759_1、9049-9759_2、 9820-9823_1's and 9820-9823_2 is amino acid modified.In some embodiments, the construct is comprising selected from this paper's Form SMCA design 9327-6054_1,9815-9825_1,9815-9825_2,9587-9735_1,9587-9735_2, 3522_1,3522_2,3519_1 and 3519_2's is amino acid modified.

In some embodiments, H1 and/or H2 is not comprising amino acid modified at the Q179 of position.In some embodiments In, H1 does not include amino acid modified Q179K not comprising amino acid modified Q179E and/or H2.In some embodiments, L1 is not Comprising amino acid modified at the S131 of position.In one embodiment, L1 does not include amino acid modified S131K.In some realities In applying scheme, L2 is not comprising amino acid modified at the T180 of position.In one embodiment, L2 is not comprising amino acid modified T180E.In some embodiments, the construct does not include such amino acid modified combination, and wherein H1 includes Q179E, L1 Comprising S131K, H2 includes Q179K, and L2 includes T180E.

In some embodiments, H1 is not comprising amino acid modified at position Q39 and/or Q179.In some embodiment party In case, H1 does not include amino acid modified Q39E and/or Q179E.In some embodiments, L1 is not comprising the ammonia at the Q160 of position Base acid modification.In one embodiment, L1 does not include amino acid modified Q160K.In some embodiments, H2 does not include position Put amino acid modified at Q179.In one embodiment, H2 does not include amino acid modified Q179K.In some embodiments In, L2 is not comprising amino acid modified at position Q38, Q160 and/or T180.In one embodiment, L2 does not include amino Acid modification Q38E, Q160E and/or T180E.In some embodiments, the construct does not include such amino acid modified group Close, wherein H1 includes Q160K comprising Q39E and/or Q179E, L1, H2 includes Q179K, and L2 comprising Q38E, Q160E and/or T180E.For example, in some embodiments, the construct does not include such amino acid modified combination, wherein：I () H1 is included Q179E, L1 include Q160K, and H2 includes Q179K, and L2 includes Q160E and T180E；(ii) H1 includes Q39E and Q179E, L1 Comprising Q160K, H2 includes Q179K, and L2 includes Q38E, Q160E and T180E；Or (iii) H1 includes Q39E, L1 is included Q160K, H2 include Q179K, and L2 includes Q38E, Q160E and T180E.

In some embodiments, H1 is not comprising amino acid modified at the Q179 of position.In some embodiments, H1 is not Comprising amino acid modified Q179K or Q179E.In some embodiments, L1 is not comprising the amino at position Q160 and/or T180 Acid modification.In one embodiment, L1 does not include amino acid modified Q160E, Q160K and/or T180E.In some embodiment party In case, H2 is not comprising amino acid modified at the Q179 of position.In one embodiment, H2 does not include amino acid modified Q179K Or Q179E.In some embodiments, L2 is not comprising amino acid modified at position Q160 and/or T180.In an embodiment party In case, L2 does not include amino acid modified Q160K, Q160E and/or T180E.In some embodiments, the construct does not include Such amino acid modified combination, wherein H1 includes Q160E, Q160K and/or T180E, H2 bags comprising Q179K or Q179E, L1 Containing Q179K or Q179E, and L2 includes Q160K, Q160E and/or T180E.

In some embodiments, H1 and/or H2 is not comprising amino acid modified at the Q179 of position.In some embodiments In, H1 does not include amino acid modified Q179E not comprising amino acid modified Q179K and/or H2.In some embodiments, L1 is not Comprising amino acid modified at the T180 of position.In one embodiment, L1 does not include amino acid modified T180E.In some realities In applying scheme, L2 is not comprising amino acid modified at the S131 of position.In one embodiment, L2 is not comprising amino acid modified S131K.In some embodiments, the construct does not include such amino acid modified combination, and wherein H1 includes Q179K, L1 Comprising T180E, H2 includes Q179E, and L2 includes S131K.

In some embodiments, H1 is not comprising amino acid modified at the Q179 of position.In some embodiments, H1 is not Comprising amino acid modified Q179E.In some embodiments, L1 is not comprising amino acid modified at the Q160 of position.In a reality In applying scheme, L1 does not include amino acid modified Q160K.In some embodiments, H2 is not comprising the amino acid at the Q179 of position Modification.In one embodiment, H2 does not include amino acid modified Q179K.In some embodiments, L2 does not include position It is amino acid modified at T180.In one embodiment, L2 does not include amino acid modified T180E.In some embodiments, The construct does not include such amino acid modified combination, and wherein H1 includes Q179E, and L1 includes Q160K, and H2 includes Q179K, and And L2 includes T180E.

In some embodiments, H1 is not comprising amino acid modified at the A139 of position.In some embodiments, H1 is not Comprising amino acid modified A139C.In some embodiments, L1 is not comprising amino acid modified at the F116 of position.In a reality In applying scheme, L1 does not include amino acid modified F116C.In some embodiments, the construct does not include such amino acid Modification combination, wherein H1 includes A139C, and L1 includes F116C.

In some embodiments, the construct is not comprising the natural disulphide bonds between heavy chain and light chain.For example, at some In embodiment, the cysteine at the position 214 of L1 and/or L2 is modified to another amino acid.In some embodiments In, L1 and/or L2 includes amino acid modified C214S.In some embodiments, half Guang ammonia at the position 233 of H1 and/or H2 Acid is modified to another amino acid.In one embodiment, H1 and/or H2 includes amino acid modified C233S.

The embodiment described herein is applied to the construct of Fab forms and whole antibody form.

Brief description

Fig. 1 show the variable region of D3H44 heavy chains and light-chain amino acid sequence and typical human germline sequence, constant region and J areas fragment is compared and (annotated in figure：* sequence iden).Figure 1A shows that (each family shows a generation to people's VH germline subgroups Table sequence).Sequence iden is compared based on D3H44 and VH3's and IGHJ3*02.Figure 1B shows people's κ VL germline subgroups (each family shows a representative series).Sequence iden is compared based on D3H44 and VKI's and IGKJ1*01.Fig. 1 C show People's λ VL germline subgroups have been gone out (each family shows a representative series).Sequence iden based on D3H44 and VL1 and The comparison of IGLJ1*01.Fig. 1 D show people's CH1 allelic sequences.Fig. 1 E show people's κ and λ allelic sequences.

Fig. 2 is shown for identifying key sequence boundary residue and for being designed using preferential heavy chain-light chain pairing The flow chart of computation modeling.

Fig. 3 shows the exemplary group of H1, L1, H2, L2 chain, and they are designed such as the H1 compared with L2 and preferentially match somebody with somebody with L1 It is right, and H2 is preferentially matched with L2 compared with L1.There is provided Variable region heavy and the animation table of the 3D crystal structures at light chain interface Show.The mutation at introducing interface is realized and preferentially forms obligate electrostatic and spatial complementary to H1-L1 and H2-L2 respectively.It is another Aspect, it is incorrect to there is unfavorable space and electrostatic mispairing with centering, this pairing for causing mispairing pair is inclined to reduce and Stability is reduced.

Fig. 4 shows the engineered requirement to form bispecific Mab (monoclonal antibody) and heavy chain-light chain to quantitative institute The high-level schematic overview figure that the analysis for needing is required.High-purity (that is, H-L associate little mispairing or without mispairing) is engineered double The design requirement of specific Mab can pass through the weight of reasonably engineered (by introducing specific amino acid mutation) two uniquenesses Chain is realized with the preferential pairing of the same endogenous light chain of its uniqueness.The process schematically shows；Herein H1 by engineered for excellent First match with L1 rather than L2.Equally, H2 by engineered preferentially to match with L2 rather than L1.Bispecific Mab designs Experiment screening requires that analysis being capable of simultaneous quantitative H1-L1: H1-L2 and H2-L2: H2-L1.These analysis requirements can be double by each Specific Fab arms can be independently engineered hypothesis simplify.In this case, the analysis only needs quantitative H1-L1: H1-L2 Or H2-L2: H2-L1, without the need for carrying out quantitatively to the two simultaneously.

Fig. 5 provides the schematic diagram of the label and preferential pairing for how determining heavy chain and light chain.In the schematic diagram, circle Expression has wherein transfected the cell of 3 constructs.Expression product is secreted from cell, makes supernatant (SPNT) flow through testing equipment (SPR chips in this case).According to the detection water of two different labels for being fused to the two light chains for competing heavy chain pairing It is flat, the quantitative predication of heavy chain and the preferential pairing of two light chains can be carried out.

Fig. 6 shows box traction substation, and it illustrates the pairing of each cluster：The average LCCA performances of the Fab heterodimers of mispairing Be worth is at least 86: 14.

Fig. 7 shows A) WT Fab heterodimers and B) representative design Fab heterodimers representativeness UPLC-SEC collection of illustrative plates (LCCA designs 9735,9737 and 9740 H1L1Fab parts).

When Fig. 8 shows that two different light chains heavy chains different from two are co-expressed in cell, it is contemplated that possibility weight Chain association product.Preferential pairing is assessed using SMCA (monoclonal antibody competition analysis).

Fig. 9 show a) D3H44/ trastuzumabs, b) D3H44/ Cetuximabs (cetuximab) and c) trastuzumab/ Preferences/chain in Cetuximab dual specificity system utilizes Preference.Different types of chain is seen using assessment by LC-MS Examine.X- axles represent H1: H2: L1: L2DNA ratio, and Y- axles show the corresponding percentage of every chain in different transfection experiments.Flat In balance system, all H and L chains have 25%.The preferences that a light chain is utilized are observed in all dual specificity systems.

Figure 10 shows the representative UPLC-SEC figures of WT heterodimers and engineered heterodimer antibody Spectrum.Figure 10 a and 10b relate separately to D3H44/ trastuzumab WT and 9060-9756_1.It is western that Figure 10 c and 10d relate separately to D3H44/ Appropriate former times monoclonal antibody WT and 9820-9823_1.Figure 10 e and 10f relate separately to trastuzumab/Cetuximab WT and 9696-9848_1.

Figure 11 shows the Fab parts of correct pairing and all utilization identical heavy chains, the change hundred of the Fab parts of mispairing Divide than (H1: L1 competes D3H44/ trastuzumabs and D3H44/ Cetuximabs with all H1 species relative to wild type；H2∶L2 Trastuzumab/Cetuximab is competed with change of all H2 species relative to wild type) and required bispecific antibody The box traction substation of the change percentage of the engineered bispecific antibody sample of each cluster is competed relative to wild type.Show every The Fab parts of the correct pairing of individual system and all utilization identical heavy chains and cluster, the change percentage of the Fab parts of mispairing, Wherein a) D3H44/ trastuzumabs, c) D3H44/ Cetuximabs and e) trastuzumab/Cetuximab.Show each system Required bispecific antibody relative to wild type and cluster change percentage, wherein b) D3H44/ trastuzumabs, d) D3H44/ Cetuximabs and f) trastuzumab/Cetuximab.In all dual specificity systems, the Fab portions of correct pairing Part is shown in Figure 11 g with all utilization identical heavy chains and cluster, the change percentage of the Fab parts of mispairing, and required is double special Property antibody be shown in Figure 11 h relative to the change percentage of wild type and cluster.It is noted that the value reported is also including engineered Bispecific antibody sample estimated change, wherein corresponding wild type construct not by SMCA assess.

Figure 12 show use provided herein is obligate mutation method that bispecific antibody is prepared to library.

Describe in detail

There is provided herein antigen-binding polypeptides construct (also referred to as heterodimer to), the construct can be different comprising first Source dimer and the second heterodimer, wherein each heterodimer include heavy chain immunoglobulin or its fragment and immune ball Protein light chain.Two heterodimers can be comprising one or more amino in heavy chain immunoglobulin constant domain 1 (CH1) One or more in acid modification and immunoglobulin light chain constant domain (CL) are amino acid modified；Heavy chain immunoglobulin can One or more in structure changes domain (VH) are amino acid modified and immunoglobulin light chain variable domains (VL) in one or It is multiple amino acid modified；Or aforementioned amino acid modification and heavy chain and the constant and variable domains combination of light chain.Jing is repaiied The amino acid of decorations is typically the part at the interface between light chain and heavy chain, and modified generating each heavy chain and required Preferential pairing between light chain so that the heavy chain of the first heterodimer is preferentially matched somebody with somebody with a light chain rather than another light chain It is right.Equally, the heavy chain of the second heterodimer can be matched preferentially with the second light chain rather than the first light chain.

As described above, to contribute to heavy chain preferential with specific light chain for amino acid modified particular combination as herein described Pairing so that bispecific monoclonal antibody (Mab) is expressed with negligible or limited mispairing, and make to never need to or Heterodimer needed for the product purification of mispairing need minimize.Heterodimer can be shown equivalent to not including amino The heat endurance of the heterodimer of acid modification, also can be shown that equivalent to not including the anti-of amino acid modified heterodimer Former binding affinity.The design of the first and second heterodimers can be used to generate two different therapeutic targets of targeting or targeting The bispecific antibody of two different epitopes (overlapping or non-overlapped) in same antigen.

The preparation method of heterodimer pair is also provided herein.

Definition

Unless otherwise defined, all technologies used herein and scientific terminology have and claimed theme art The identical meanings that are generally understood of technical staff.If the term of this paper there are multiple definition, it is defined by this part.Work as reference When URL or other this class identifiers or address, it should be understood that this class identifier can change, and the specifying information on internet is continuous Change, but the information being equal to can be obtained by searching for Internet.This type of information is quoted and confirms its availability and open propagation.

It should be appreciated that above-mentioned general description and detailed description below be merely exemplary with it is explanatory, and not It is limited to claimed any theme.In the present patent application, unless otherwise specified, the use of odd number also includes plural number.

In the specific embodiment of the present invention, any concentration range, percentage range, ratio ranges or integer range should It is interpreted as including any integer value in listed scope, and (as being suitable for) their fraction (1/10th Hes of such as integer One of percentage), except as otherwise noted.As used herein, " about " ± the 10% of indicating range, value, sequence or structure is meant, unless It is otherwise indicated.It should be appreciated that as used herein, term " one " and " one kind " refer to " one or more " in listed component, Unless context is otherwise indicated or requires.Substitute word (for example, "or") use be understood to mean that one of alternative form, two Person or any combination of them.As used herein, term " including " and "comprising" can be used synonymously.Additionally, should manage Solution, this patent application discloses from the single single chain polypeptide of the various combinations of structure as herein described and substituent or immune ball Protein constructs, to reach the separately shown degree of each single chain polypeptide or heterodimer is seemed.Therefore, form single single-stranded The selection of the concrete component of polypeptide or heterodimer is in the scope of the present disclosure

Subhead used herein is only used for organizational goal, and should not be construed as limiting described theme.This patent Shen The all documents that please be quote or the part of document, including but not limited to patent, patent application, article, books, handbook and discussion evidence This is clearly incorporated by reference in its entirety for any purpose.

It should be appreciated that method described herein and composition be not limited to concrete grammar as herein described, scheme, clone, Construct and reagent, therefore can be change.It is also understood that term used herein is only used for describing specific embodiment Purpose, and be not intended to limit the scope of method described herein and composition, the scope is only entered by claims Row is limited.

The all disclosures being mentioned above and full patent texts are herein incorporated by reference for describing and disclosing purpose, for example Construct and method described in open can be used with reference to method described herein, composition and compound.The public affairs being discussed herein Open and be provided separately, to carry out disclosure before the submission date of present patent application.Should not be understood herein be according to aforementioned invention or For any other reason, it is allowed to which inventor as herein described understands in advance such disclosure.

In the present patent application, the use such as Protein of amino acid name and atomic name (such as N, O, C etc.) DataBank (PDB) (www.pdb.org) is defined, and these titles are based on IUPAC nomenclatures (IUPAC Nomenclature And Symbolism for Amino Acids and Peptides (the IUPAC nomenclatures and symbol (residue of amino acid and peptide Title, atomic name etc.)), Eur.J.Biochem., 138,9-37 (1984) and its correction Eur.J.Biochem., 152,1 (1985)).Term " amino acid residue " be directed primarily to represent by 20 kinds of naturally occurring amino acid, i.e. alanine (Ala or A), Cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly Or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), silk ammonia Sour (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y) residue group Into group in the amino acid residue that includes.

Term " polypeptide ", " peptide " and " protein " is used interchangeably herein, and refers to the polymer of amino acid residue.I.e. The description for being related to polypeptide is equally applicable to the description of peptide and the description of protein, and vice versa.The term is applied to naturally occurring Amino acid polymer and wherein one or more amino acid residues be non-naturally encoded amino acid amino acid polymer. As used herein, the term covers the amino acid chain of any length, is connected by covalent peptide bonds including wherein amino acid residue Full length protein.

Term " nucleotide sequence " or " nucleotide sequence " are intended to indicate that the continuous fragment of two or more nucleic acid molecules. Nucleotide sequence can be genome, cDNA, RNA, semi-synthetic or synthesis source or any combination of them.

" cell ", " host cell ", " clone " and " cell culture " is used interchangeably herein, and it is all this Class term is understood to include the filial generation that cell growth or culture are produced." conversion " and " transfection " is used interchangeably, and refers to core Acid sequence introduces the process of cell.

Term " amino acid " refers to naturally occurring and non-naturally occurring amino acid, and with similar to naturally occurring The amino acid analogue and amino acid simulant of the mode function of amino acid.The amino acid of natural coding is 20 kinds common Amino acid (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, group ammonia Acid, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, junket ammonia Acid and valine) and pyrrolysine and selenocystein.Amino acid analogue refers to have with naturally occurring amino acid The compound of identical basic chemical structure, i.e. carbon are bound to hydrogen, carboxylic group, amino group and R bases, such as, homoserine, Nor-leucine, methionine sulfoxide, methionine methyl sulfonium.Such analog has modified R bases (such as, nor-leucine) Or modified peptide backbone, but keep and naturally occurring amino acid identical basic chemical structure.The reference of amino acid includes Such as naturally occurring proteinacious l-amino acid；The amino acid D- amino acid of Jing chemical modifications, such as amino acid variant and spread out It is biological；Naturally occurring nonprotein acidic amino acid, alanine, ornithine etc.；And with known in the art as amino The compound of the chemical synthesis of the property of sour feature.The example of non-naturally occurring amino acid includes but is not limited to-methylamino Sour (such as methylalanine), D- amino acid, histidine sample amino acid (for example, 2- amino-histidines, hydroxy-histidine, height Histidine), there is in side chain the amino acid (" height " amino acid) and the carboxylic acid functional wherein in side chain of other methylene The amino acid replaced by sulfonic acid group (for example, cysteic acid).By non-natural amino acid (including the non-natural amino of synthesis Acid, displacement amino acid or one or more D- amino acid) mix in a multitude of different ways the present invention protein can be favourable 's.Amino acid peptide containing D- etc. shows more external than the homologue containing l-amino acid peptide or internal stability to be increased.Therefore, expectation is worked as Or when needing bigger intracellular stability, the structure for mixing the peptide of D- amino acid etc. can be useful especially.More particularly, D- peptides etc. are the peptase of resistance to endogenous and protease, so as to when such property is expected, there is provided the molecular biosciences of improvement is utilized Rate simultaneously extends volume lifetime.In addition, the presentation limited for II classes MHC is to helper cell, D- Peptide etc. can not be processed effectively, therefore, HI can not possibly be caused in whole organism.

Amino acid is herein by IUPAC-IUB biochemical nomenclature commission (IUPAC-IUB Biochemical Nomenclature Commission) recommend well known three letter symbols or represented by a letter character.Equally, nucleosides The one-letter code that acid can pass through to accept extensively is represented.

" Jing guards the variant of modification " is applied to both amino acid and nucleotide sequence.For specific nucleotide sequence, " Jing The variant of conservative modification " refers to such nucleic acid：Its coding identical or substantially the same amino acid sequence, or the wherein core Sour not encoding amino acid sequence, substantially the same sequence.Due to the degeneracy of genetic code, many function identical nucleic acid are compiled Any protein specified of code.For example, the equal encoding alanine amino acid of codon GCA, GCC, GCG and GCU.Therefore, wherein Each position for the alanine that codon is specified, can be changed to any corresponding codon by codon, and does not change and compiled The polypeptide of code.Such nucleic acid modification is " silence modification ", is a species of the modification that Jing guards modification.This paper coded polypeptides Each nucleotide sequence also describes each possible silence modification of nucleic acid.One of ordinary skill in the art will be recognized that, core (in addition to AUG and TGG, AUG is typically the unique codon of methionine to each codon in acid, and TGG is typically tryptophan Unique codon) can modify and obtain function identical molecule.Therefore, each silence modification of the nucleic acid of coded polypeptide is implicitly included in In each described sequence.

For amino acid sequence, one of ordinary skill in the art will be recognized that, nucleic acid, peptide, polypeptide or protein sequence Single displacement, disappearance or the increase for arranging (it changes, increases or delete single amino acids or a small amount of amino acid in coded sequence) It is " variant that Jing guards modification ", the wherein change causes the disappearance of amino acid, the increase of amino acid or is by amino acid replacement The amino acid being chemically similar.Conservative substitution table provides functionally similar amino acid, and the table is the ordinary skill of this area Known to personnel.Such Jing guards the variant of modification also includes and is not excluded for homologue between Polymorphic variant, the species of the present invention And allele.

Conservative substitution table provides functionally similar amino acid, and the table is known to persons of ordinary skill in the art. Hereinafter each in eight groups is comprising the amino acid as mutual conservative substitution：

Alanine (A), glycine (G)；

Aspartic acid (D), glutamic acid (E)；

Asparagine (N), glutamine (Q)；

Arginine (R), lysine (K)；

Isoleucine (I), leucine (L), methionine (M), valine (V)；

Phenylalanine (F), tyrosine (Y), tryptophan (W)；

Serine (S), threonine (T)；And

Cysteine (C), methionine (M)

(see, for example, Creighton, Proteins：Structures and Molecular Properties(W H Freeman&Co.；Second edition (in December, 1993).

In the linguistic context of two or more nucleic acid or peptide sequence, term " identical " or percentage " homogeneity " refer to phase Two or more sequences together or subsequence.When carrying out maximum corresponding comparing and align in comparison window or the area for specifying When, if the percentage of the amino acid residue of sequence or nucleotides is identical, the sequence is " substantially the same " (that is, with finger Fixed area have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%th, 95%, 96%, 97%, 98% or 99% homogeneity), such as using one of following sequence comparison algorithm (or this area Available other algorithms of those of ordinary skill) or manually align and visually inspect determined.This definition is directed to test The complementary series of sequence.Homogeneity may be present in the area that length is at least about 50 amino acid or nucleotides, or length is 75- The area of 100 amino acid or nucleotides, or in the case of not specified, on whole polynucleotides or peptide sequence.Code book The polynucleotides of the polypeptide (including the homologue from species in addition to a person) of invention can pass through the method for comprising the following steps Obtain：Using the label probe screening library of the polynucleotide sequence or its fragment with the present invention under stringent hybridization condition, And separation full-length cDNA and the genomic clone comprising the polynucleotide sequence.Such hybridization technique is that technical staff knows 's.

If the amino acid sequence of the derivative of polypeptide or variant has at least with 100 amino acid sequences of initial peptide 50% homogeneity, then the derivative or variant are considered as with peptide total " homology " or are " homologous " with peptide.In some enforcements In scheme, the quantity and derivative identical peptide or fragment at least 75% phase of peptide of the derivative or variant and amino acid residue Together.In certain embodiments, the quantity of the derivative or variant and amino acid residue and derivative identical peptide or the piece of peptide Section at least 85% is identical.In certain embodiments, the amino acid sequence of the derivative and the quantity of amino acid residue with it is derivative The fragment of thing identical peptide or peptide at least 90% is identical.In some embodiments, the amino acid sequence and amino of the derivative The quantity of sour residue is identical with the fragment at least 95% of derivative identical peptide or peptide.In certain embodiments, the derivative Or variant is identical with the fragment at least 99% of derivative identical peptide or peptide with the quantity of amino acid residue.

As used herein, " detached " polypeptide or construct mean to identify and divide from the component of its n cell culture environment From and/or reclaim construct or polypeptide.The pollution components of its natural surroundings are generally to hinder the diagnosis of heteromultimers or control The material of purposes is treated, and may include enzyme, hormone and other proteinacious or non-proteinaceous solute.

In certain embodiments, as used herein, " detached " antigen-binding polypeptides construct as herein described includes Heterodimer pair or " detached " heterodimer pair, are identified and isolated from including the component from its n cell culture environment And/or the heterodimer or heterodimer pair of recovery.The pollution components of its natural surroundings are to hinder heterodimer or anti- The diagnosis of former Binding peptide construct or the material of therapeutical uses, and may include enzyme, hormone and other proteinacious or non-egg White matter solute.

Heterodimer and antigen-binding polypeptides construct and heterodimer are substantially homogeneous to being generally purified to.Phrase " substantially homogeneous ", " substantially homogeneous form " and " substantially homogeneous " is substantially free of from undesirable for expression Polypeptides in combination (such as homodimer) accessory substance product.In the linguistic context, species of interest is comprising H1 and L1 (H1-L1) or H2 and L2 (H2-L2) heterodimer.Pollutant is included comprising H1 and L2 (H1-L2) or H2 and L1 (H2-L1) Heterodimer or comprising H1 and L1 or H2 and L2 (no matter Fab parts are correct pairing or mispairing).With regard to purity Speech, the substantially homogeneous amount for meaning accessory substance is less than 10%, such as total LC-MS intensity of all kinds present in mixture Less than 5%, less than 1% or less than 0.5%, wherein the percentage reflects the result of mass spectral analysis.

Phrase " selective (or specificity) is hybridized to " is referred to when the sequence is present in compound mixture (including but not limited to Total cell or library DNA or RNA) in when, under stringent hybridization condition molecule only combined with specific nucleotide sequence, in pairs or Hybridization.

The term that the technical staff in antibody technique field understands, each is respectively provided with the implication of this area imparting, unless at this Clearly there is different definition in text.Known antibodies have variable region, hinge area and constant domain.Immunoglobulin structure Compile in such as Harlow etc. with function, Antibodies：A Laboratory Manual, the 14th chapter (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988) in commented.

As used herein, term " antibody " and " immunoglobulin (Ig) " or " antigen-binding polypeptides construct " are used interchangeably. " antigen-binding polypeptides construct " is referred to substantially by one or more immunoglobulin genes, or their one or more pieces The polypeptide of section coding, polypeptid specificity ground bound analyte (antigen).Generally acknowledged immunoglobulin gene include κ, λ, α, γ, δ, ε and μ constant region gene, and countless immune globulin variable region genes.Light chain is classified as κ or λ.Heavy chain is classified as γ, μ, α, δ or ε, define respectively Immunoglobulin Isotype IgG, IgM, IgA, IgD and IgE then.In addition, antibody can belong to One of multiple hypotypes, for example, IgG can belong to IgG1, IgG2, IgG3 or IgG4 subclass.

Exemplary immunoglobulin (antibody) construction unit is made up of two pairs of polypeptide chains, and each pair has " light " chain (about 25kD) with " weight " chain (about 50-70kD).Term " light chain " is included with enough variable region sequences to give with reference to special The full-length light chains and its fragment of property.Full-length light chains include Variable domain VL and constant region domain CL.The variable region of light chain Domain is located at the amino terminal of polypeptide.Light chain includes κ chains and λ chains.Term " heavy chain " is included with enough variable region sequences To give the total length heavy chain and its fragment of binding specificity.Total length heavy chain includes Variable domain VH and three constant region domains Domain CH1, CH2 and CH3.VH domains be located at polypeptide amino terminal, CH domains be located at carboxyl terminal, wherein CH3 near The carboxyl terminal of polypeptide.Heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subclass), IgA (including IgA1 and IgA2 subclass), IgM and IgE.Term " variable region " or " variable domains " refer to a part of light chain of antibody And/or heavy chain, be generally responsible for antigen recognizing, generally include 120 to 130 amino acid of amino terminal in about heavy chain (VH) and About 100 to 110 amino terminal amino acids in light chain (VL).It is special that " complementary determining region " or " CDR " contributes to antigen binding The amino acid sequence of the opposite sex and affinity." framework " area (FR) can help to maintain the appropriate conformation of CDR, to promote antigen binding Combination between area and antigen.In structure, framework region can be located at the antibody between CDR.Variable region typically exhibits identical With respect to the formula of conserved framework regions (FR), the framework region is by three hypervariable region CDR connections.The CDR of two chains of each pair Generally alignd by framework region, the framework region can allow to be combined with defined epitope.From N- ends to C- ends, light chain and heavy chain can Become both areas and generally comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domain.The Amino acid score of each domain is matched somebody with somebody Generally according to Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)) definition carry out, except as otherwise noted.At certain In a little embodiments, antigen-binding polypeptides construct is included and is connected to IgG, IgM, IgA, IgD or IgE for treating polypeptide at least One immunoglobulin domains.In some embodiments, provided herein is exempting from of including of antigen-binding polypeptides construct Epidemic disease imrnuglobulin domain is from the construct based on immunoglobulin (Ig), such as double antibody or monomer.In certain embodiments, originally Antigen-binding polypeptides construct described in text is tied comprising at least one immunoglobulin (Ig) from heavy chain antibody such as alpaca antibody Structure domain.In certain embodiments, provided herein is antigen-binding polypeptides construct include from mammalian antibody such as ox Antibody, human antibody, alpaca antibody (single domain and non-single domain), rodent antibodies, humanized antibody, non-humanization resist At least one immunoglobulin domains of body, mouse antibodies or any chimeric antibody.In certain embodiments, provided herein is Antigen-binding polypeptides construct comprising from synthetic library generate antibody at least one immunoglobulin domains.

" bispecific ", " dual specificity " or " difunctional " antigen-binding proteins or antibody be have two it is different anti- The hybrid antigens associated proteins of former binding site.Bispecific antigen binding proteins and antibody are a kind of polyspecific antigen bindings Protein antibodies species.It is somebody's turn to do with reference to two different epi-positions in the two basic change site of Bispecific antigen binding proteins or antibody Epi-position may be present in identical or different molecular target." polyspecific antigen-binding proteins " or " multi-specificity antibody " are targetings More than an antigen or the antibody of epi-position." bivalent antigen-binding protein " or " bivalent antibody " includes two antigen binding sites. In some cases, two basic change site has identical antigentic specificity.Bivalent antigen-binding protein and bivalent antibody can be with It is bispecific, see below.In certain embodiments, in addition to " polyspecific " or " multi-functional " antibody divalence resists It is identical that body is generally understood as each binding site.

Term " preferential pairing " is used for herein the matching method of the first polypeptide of description and the second polypeptide, such as antigen knot Close the heavy chain immunoglobulin and light chain immunoglobulin of polypeptide construct and heterodimer centering as herein described.Therefore, " preferential pairing " refers to one or more other not homopolypeptide of presence while pairing between the first and second polypeptides When, the preferred pairing of the first polypeptide and the second polypeptide.Generally preferential pairing be one of first and second polypeptides or The result of the modification (for example, amino acid modified) of the two.Generally, preferential pairing produces the pairing of the first and second polypeptides, is pairing The most abundant dimer existed after generation.It is known in the art, if heavy chain immunoglobulin (H1) immunity different from two Immunoglobulin light chains (L1 and L2) are co-expressed, and will match with two light chains with statistically waiting probability, produce H1 and L1 match and About 50: 50 mixtures of H1 and L2 pairings.In the linguistic context, when H1 and both L1 and L2 are co-expressed, if H1-L1 weights The amount of chain-light chain heterodimer more than H1-L2 heterodimers amount, " preferential pairing " will occur in such as H1 and L1 it Between.Therefore, in this case, H1 is preferentially matched with L1 compared with L2.

However, generating in the linguistic context of wild type bispecific antibody from two starting antibody systems, it is also known in the art, In some cases, when the light chain of an antibody forming system is preferentially matched with the heavy chain of two antibody forming systems, exist intrinsic inclined Love property.Therefore, when in the linguistic context in bispecific antigen-binding constructs determine design strength when, with wild type system in match somebody with somebody Degree is compared, the pairing degree for assessing the design can be necessary.Therefore, in one embodiment, if required is double More than the amount of the bispecific antibody needed for obtaining in wild type system, then design is considered as showing the amount of specific antibody Go out preferential pairing.In another embodiment, if in the weaker arm of antibody, the amount of pairing is more than in wild type system deposits Amount, then design is considered as showing preferential pairing.

Heavy chain of antibody is matched with light chain of antibody, and is joined each other or contacted at one or more " interfaces "." interface " wraps Include one or more " contact " amino acid residues of the first polypeptide, one or more of the residue and the second polypeptide " contact " ammonia Base acid residue interacts.For example, interface is present between the CH3 peptide sequences of dimerization CH3 domains, the CH1 of heavy chain is tied Between structure domain and the CL domains of light chain and between the VH domains of heavy chain and the VL domains of light chain." interface " can derive from IgG antibody, such as from human IgG1's antibody.

As used herein, term " amino acid modified " includes but is not limited to amino acid mutation, insertion, disappearance, displacement, chemistry Modification, physical modification and rearrangement.

Antigen-binding polypeptides construct and heterodimer pair

Antigen-binding polypeptides construct as herein described can be comprising the first heterodimer and the second heterodimer；Each Heterodimer is obtained by heavy chain immunoglobulin with the pairing of light chain immunoglobulin.Heavy chain immunoglobulin and light chain The structure and tissue of constant and variable domains is well known in the art.Heavy chain immunoglobulin is generally comprised one variable (VH) Domain and three constant domains CH1, CH2 and CH3.Light chain immunoglobulin generally comprise variable (VL) domain and One constant (CL) domain.Various modifications can be made to these typical forms.

Antigen-binding polypeptides construct as herein described and heterodimer pair can include the first heterodimer and second Heterodimer, each heterodimer includes the immunoglobulin (Ig)/heavy chain of antibody or its piece with least VH and CH1 domains Section, and the immunoglobulin (Ig)/light chain of antibody with VL domains and CL domains.In one embodiment, heterodimeric Two heterodimers of body pair and antigen-binding polypeptides construct include full-length immunoglobulin heavy chain.In another embodiment party In case, two heterodimers of heterodimer pair or antigen-binding polypeptides construct comprising heavy chain immunoglobulin (including At least VH and CH1 domains) fragment.In one embodiment, two heterodimers of heterodimer pair are included and exempted from The amino end segment of epidemic disease immunoglobulin heavy chain (comprising at least VH and CH1 domains).In another embodiment, heterodimeric Carboxy terminal fragment of two heterodimers of body pair comprising heavy chain immunoglobulin (comprising at least VH and CH1 domains).

Each heterodimer of heterodimer pair can be specifically bound to antigen or epi-position.In an embodiment In, the heavy chain immunoglobulin and light chain immunoglobulin of each heterodimer are derived from the one of known antibodies such as treatment antibody It is individual or it is multiple modification or by its engineering.Treatment antibody is for suffering from or tending to the mammal with disease or illness The antibody of middle treatment disease or illness.It is mono- that the suitable treatment antibody in each heterodimer source includes but is not limited to A Bafu Anti- (abagovomab), adalimumab (adalimumab), alemtuzumab (alemtuzumab), aurograb, bar pearl list Anti- (bapineuzumab), basiliximab (basiliximab), Baily wood monoclonal antibody (belimumab), bevacizumab (bevacizumab), mine-laying slave monoclonal antibody (briakinumab), block that monoclonal antibody (canakinumab), catumaxomab (catumaxomab), Pegylation plug trastuzumab (certolizumab pegol), Cetuximab, daclizumab (daclizumab) promise monoclonal antibody (denosumab), efalizumab (efalizumab), galiximab, (galiximab), wooden monoclonal antibody (golimumab) in lucky trastuzumab Ao Jia meter stars (gemtuzumab ozogamicin), dagger-axe, Ibritumomab tiuxetan (ibritumomab tiuxetan), infliximab (infliximab), according to a wooden monoclonal antibody (ipilimumab), Shandong former times monoclonal antibody (lumiliximab), mepolizumab (mepolizumab), dimension pearl monoclonal antibody (motavizumab), muromonab (muromonab), mycograb, natalizumab (natalizumab), Buddhist nun's trastuzumab (nimotuzumab), auspicious pearl monoclonal antibody (ocrelizumab) difficult to understand, difficult to understand (ofatumumab), Ao Mazuo monoclonal antibodies (omalizumab), palivizumab (palivizumab), Victibix (panitumumab), handkerchief trastuzumab (pertuzumab), thunder Buddhist nun pearl monoclonal antibody (ranibizumab), Rayleigh pearl monoclonal antibody (reslizumab), Rituximab (rituximab), Mortopl profit pearl monoclonal antibody (teplizumab), Tosi pearl monoclonal antibody (tocilizumab/atlizumab), Tosi not Monoclonal antibody (tositumomab), trastuzumab (trastuzumab), general sieve former times ammonium (ProxiniumTM), RencarexTM, excellent spy Gram monoclonal antibody (ustekinumab) and prick Shandong mesh monoclonal antibody (zalutumumab).

In one embodiment, the heavy chain immunoglobulin of each heterodimer and/or light chain immunoglobulin source The one or more modifications of the antibody of self-bonding molecule are engineered by it, and the molecule includes but is not limited to following protein row Table, and belong to subunit, domain, motif and the epi-position of following protein list：Feritin；Growth hormone, including people growth swash Element and BGH；Somatotropin releasing factor；Parathyroid hormone；Thyrotropic hormone；Lipoprotein；The anti-tryptoses of α -1- Enzyme；INSULIN A-chain；Insulin B-chain；Proinsulin；Follicle-stimulating hormone (FSH)；Calcitonin；Luteinising hormone；Glucagons；Blood coagulation The factor such as factor Ⅴ II, Factor IX C, factors IX, tissue factor (TF) and the temperature Wei Baishi factors；Anticoagulin such as egg White C；The atrial natriuretic factor；Curosurf；Plasminogen activator, such as urokinase or human urine or tissue Type plasminogen activator (t-PA)；Bell toad element；Fibrin ferment；Hemopoieticgrowth factor；Tumor necrosis factor-alpha and-β； Enkephalinase；RANTES (the normal T-cell expression adjusted during activation and EF)；Human macrophage inflammatory protein (MIP- 1-α)；Seralbumin such as human serum albumins；MIS；Relaxain A- chains；Relaxain B- chains；Relaxain It is former；Little mouse promoting sexual gland hormone related peptide；Microprotein, such as beta-lactamase；DNA enzymatic；IgE；Born of the same parents' poison T- lymphocytes Related antigen (CTLA), such as CTLA-4；Inhibin；Activin；VEGF (VEGF)；Hormone or growth factor Acceptor, such as EGFR, VEGFR；Interferon such as IFN-α (α-IFN), IFN-β (β-IFN) and IFN-γ (γ-IFN)；Albumin A or D；Rheumatoid factor；Neurotrophic factor such as bone derived neurotrophic factor (BDNF), nerve battalion Foster protein-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6) or nerve growth factor；Platelet derived growth factor (PDGF)；Fibroblast growth factor such as AFGF and PFGF；EGF (EGF)；TGF (TGF) is all Such as TGF- α and TGF-β, including TGF-1, TGF-2, TGF-3, TGF-4 or TGF-5；Insulin like growth factor-1 and-II (IGF-I and IGF-II)；Des (1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein；CD albumen is such as CD2、CD3、CD4、CD8、CD11a、CD14、CD18、CD19、CD20、CD22、CD23、CD25、CD33、CD34、CD40、 CD40L, CD52, CD63, CD64, CD80 and CD147；Hematopoietin；Osteogenesis induction factor；Immunotoxin；Bone shape There is albumen (BMP) in state；Interferon such as interferon-' alpha ' ,-β and-γ；Colony stimulating factor (CSF), such as M-CSF, GM-CSF And G-CSF；Interleukin (IL), such as IL-1 to IL-13；TNF-α, erythrocuprein；T-cell receptors；Surface membrane protein； Decay accelerating factor；Viral antigen, such as a part for AIDS coatings, such as gp120；Transport protein；Homing receptor；Ground Location element；Regulatory protein；Cell adhesion molecule such as LFA-1, Mac 1, p150.95, VLA-4, ICAM-1, ICAM-3 and VCAM, A4/p7 integrins and (Xv/p3 integrins include or its subunit, integrin alpha subunit such as CD49a, CD49b, CD49c, CD49d, CD49e、CD49f、α7、α8、α9、αD、CD11a、CD11b、CD51、CD11c、CD41、αIIb、αIELb；Integrin beta subunit is all Such as CD29, CD 18, CD61, CD104, β 5, β 6, β 7 and β 8；Integrin subunit combination includes but is not limited to α V β 3, α V β 5 and the β of α 4 7；The member of apoptotic pathways；IgE；Blood group antigens；Flk2/flt3 acceptors；Fat (OB) acceptor；Mp1 acceptors；CTLA-4； PROTEIN C；Eph acceptor EphA2, EphA4, EphB2 etc.；Human leucocyte antigen (HLA) (HLA) such as HLA-DR；Complement protein is such as Complement receptor CRI, C1Rq and other complement factors such as C3 and C5；Glycoprotein receptor such as GpIb. α., GPIIb/IIIa and CD200；And the fragment of any of above polypeptide listed.

In one embodiment, the heavy chain immunoglobulin and/or light chain of each heterodimer is derived from specificity knot Close the one or more modifications of the antibody of cancer antigen or by its engineering, the antibody includes but is not limited to ALK acceptors, and (multiple-effect is given birth to Growth factor receptor body), multiple effect growth factor, the pancarcinoma antigens of KS 1/4；OCA (CA125)；Prostatic acid phosphatase；Prostatitis Gland specific antigen (PSA)；Melanoma associated antigen p97；Melanoma antigen gp75；HMW melanoma antigen (HMW- MAA)；PSMA；Carcinomebryonic antigen (CEA)；Polymorphic epithelial mucin antigen；HMFG's antigen；Knot Intestines rectal neoplasm related antigen is such as：CEA, TAG-72, C017-1A, GICA 19-9, CTA-1 and LEA；Burkitt lymphoma resists Former -38.13；CD19；People B- lymph tumor antigen-CD20；CD33；Melanoma specific antigens such as gangliosides GD2, nerve Section glycosides fat GD3, Ganglioside GM2 and Ganglioside GM3；Tumor Specific Transplantation type cell surface antigen (TSTA)；Disease The tumour antigen that poison causes, including the T- antigens and the envelope antigen of RNA tumour viruses of DNA tumour viruses；Carcinomebryonic antigen first tire The CEA of albumen such as colon, 514 cancer embryo trophoderm glycoprotein and tumor of bladder carcinomebryonic antigen；Differentiation antigen such as human lung cancer resists Former L6 and L20；The antigen of fibrosarcoma；Leukemia T cells antigen-Gp37；Neoglycoproteins；Sphingolipid；Breast cancer resists Original such as EGFR (EGF-R ELISA)；NY-BR-16；NY-BR-16 and HER2 antigens (p185HER2)；Polymorphism epithelium Mucoprotein (PEM)；Pernicious human lymphocyte antigen-APO-1；Differentiation antigen, is such as found in the I antigen of FE；Deposit It is the primary endoblast I antigen of adult erythrocyte；PIE；It is present in the I (Ma) of sdenocarcinoma of stomach；It is present in breast epithelium M18、M39；It is present in the SSEA-1 of bone marrow cell；VEP8；VEP9；Myl；Va4-D5；It is present in the D156- of colorectal cancer 22；TRA-1-85 (blood group H)；It is present in the SCP-1 of testis and oophoroma；It is present in the C14 of adenocarcinoma of colon；It is present in adenocarcinoma of lung F3；It is present in the AH6 of cancer of the stomach；Y haptens；It is present in the Ley of embryonal carcinoma cell；TL5 (blood group A)；It is present in A431 thin The EGF receptor of born of the same parents；It is present in E1 series (blood group B) of cancer of pancreas；It is present in the FC10.2 of embryonal carcinoma cell；Sdenocarcinoma of stomach resists It is former；It is present in the CO-514 (blood group Lea) of gland cancer；It is present in the NS-10 of gland cancer；CO-43 (blood group Leb)；It is present in A431 thin The G49 of the EGF receptor of born of the same parents；It is present in the MH2 (blood group ALeb/Ley) of adenocarcinoma of colon；It is present in the 19.9 of colon carcinoma；Cancer of the stomach Mucoprotein；It is present in the T5A7 of bone marrow cell；It is present in melanomatous R24；Be present in embryonal carcinoma cell 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2 and M1: 22: 25: 8 and are present in the SSEA-3 and SSEA-4 of 4 to 8 cell stage embryos； CTCL antigen；MART-1 antigens；Sialy Tn (STn) antigen；Colon cancer antigen NY-CO-45；LuCA NY-LU-12valiant A；Gland cancer antigen A RT1；Secondary tumour relevant brain-cancer testes antigens (tumprigenicity neural antigen MA2；It is secondary Tumour neuron antigen)；Neural tumor veutro antigen 2 (NOVA2)；Hepatocellular carcinoma antigen gene 520；Tumor associated antigen CO- 029；Tumor associated antigen MAGE-C1 (cancer/testis antigen CT7), MAGE-B1 (MAGE-XP antigens), MAGE-B2 (DAM6), MAGE-2, MAGE-4-a, MAGE-4-b and MAGE-X2；Cancer-testis antigen (NY-EOS-1) and it is any of above list it is many The fragment of peptide.

Human antibody can classify as isotype, including IgG, IgA, IgE, IgM and IgD.In one embodiment, Fc sources In IgG isotypes.In another embodiment, Fc derives from IgA isotypes.In another embodiment, Fc is derived from IgE isotypes.In another embodiment, Fc derives from IgM isotypes.In another embodiment, Fc derives from IgD Isotype.

Human IgG antibody can also be divided into subclass IgG1, IgG2, IgG3 and IgG4.Therefore, in some embodiments, it is contemplated that Fc can derive from IgG1, IgG2, IgG3 or IgG4 subclass of antibody.

Each heterodimer of heterodimer pair can be specifically bound to antigen or epi-position.In an embodiment In, each heterodimer of heterodimer pair combines identical epi-position.In another embodiment, heterodimer pair The first heterodimer specifically bind an antigen on epi-position, heterodimer pair the second heterodimer specificity With reference to epi-position different in same antigen.In another embodiment, the first heterodimer of heterodimer pair is special Property combine epi-position on the first antigen, the second heterodimer specific binding of heterodimer pair is different from the first antigen Epi-position on second antigen.For example, in one embodiment, the first heterodimer specific binding tissue factor, and the Two heterodimer molecule of the antigen binding Her2 (ErbB2), vice versa.In alternate embodiment, first is different Source dimer specific binding tissue factor, and the second heterodimer specific binding EGFR, vice versa.In another reality In applying scheme, the first heterodimer specific binding EGFR, and the second heterodimer molecule of the antigen binding Her2, otherwise It is as the same.In another embodiment, the first heterodimer specifically binds above-mentioned molecule or cancer antigen.In another reality In applying scheme, the second heterodimer specifically binds above-mentioned molecule or cancer antigen.

As described above, in some embodiments, the heavy chain immunoglobulin and immunoglobulin (Ig) of each heterodimer Light chain is derived from known treatment antibody or the one or more modifications with reference to various target molecules or the antibody of cancer antigen or by it Engineering.The amino acid and nucleotide sequence of many this quasi-molecules be easy to get (see, for example, GenBank：AJ308087.1 (people Source antihuman tissue factor antibody D3H44 light chain variable districts and CL domains)；GenBank：AJ308086.1 (Humanized anti-humans Tissue factor antibodies D3H44 weight chain variable districts and CH1 domains)；GenBank：HC359025.1 (handkerchief trastuzumab Fab light chains Netic module)；GenBank：HC359024.1 (handkerchief trastuzumab Fab heavy chain gene module)；GenBank：GM685465.1 is (anti- Body trastuzumab (=Herceptin (Herceptin))-wild type；Light chain)；GenBank：GM685463.1 (antibody trastuzumabs (=Herceptin)-wild type；Heavy chain)；GenBank：(it is light that antibody trastuzumab (=Herceptin)-GC optimizes GM685466.1 Chain)；And GenBank：GM685464.1 (heavy chain of antibody trastuzumab (=Herceptin)-GC optimizations)).It is as herein described every The sequence of individual GenBank numbering is available from NCBI websites, final updating on November 28th, 2012, and each by reference It is integrally incorporated for all purposes.The amino acid and nucleotide sequence of Cetuximab is also known in the art, be see, for example, By Canadian Institutes of Health Research.Alberta Innovates-Health Solutions and The Metabolomics Innovation Centre (TMIC) provides the Drug Bank websites supported, accession number DB00002。

In some respects, detached antigen-binding polypeptides construct include with shown in form disclosed herein or accession number Amino acid sequence or its fragment have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%th, the amino acid sequence of 99% or 100% homogeneity.In some respects, detached antigen-binding polypeptides construct include with Nucleotide sequence or its fragment shown in form disclosed herein or accession number have at least 80%, 85%, 90%, 91%, 92%th, the amino acid sequence of the polynucleotide encoding of 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homogeneity Row.

Heavy chain immunoglobulin and light chain it is amino acid modified

At least one of heterodimer of heterodimer pair can be comprising heavy chain immunoglobulin and/or immune globulin One or more of white light chain are amino acid modified so that the heavy chain of the first heterodimer preferentially with one of light chain rather than Other light chain pairings.Equally, the heavy chain of the second heterodimer can be matched preferentially with the second light chain rather than the first light chain.It is described One heavy chain can be based on comprising a heavy chain immunoglobulin and two immune globulins with the preferential pairing of one of two light chains The design collection (referred to as LCCA design collection) of white light chain, wherein when heavy chain immunoglobulin and two light chain immunoglobulin coexpressions When, heavy chain immunoglobulin preferentially with one of two light chain immunoglobulins rather than another one pairing.Therefore, LCCA sets Meter collection can include heavy chain immunoglobulin, the first light chain immunoglobulin and second light chain immunoglobulin.

In one embodiment, one or more are amino acid modified comprising one or more amino acid replacements.

In one embodiment, the preferential pairing that LCCA designs are set shown in is used as between light chain and heavy chain by modification Interface a part one or more amino acid setting up.In one embodiment, what LCCA designs were set shown in is excellent CH1 domains, the CL domains and second of the first light chain immunoglobulin that first pairing passes through modified immunoglobulin heavy chain One or more amino acid at least one of CL domains of light chain immunoglobulin are setting up.

In one embodiment, one or more it is amino acid modified be restricted to variable (VH, VL) and it is constant (CH1, CL) the consensus framework residue of domain, as shown in the Kabat numberings of residue.For example, Almagro [Frontiers In Bioscience(2008)13：1619-1633] determining for Framework residues is provided according to Kabat, Chotia and IMGT numbering plan Justice.

In one embodiment, at least one of heterodimer includes introducing immunoglobulin (Ig) weight complimentary to one another One or more mutation of chain and light chain immunoglobulin.The complementation of heavy chain and light chain interface can space and hydrophobic contact, Realize on the basis of the combination of electrostatic/charge interaction or various interactions.Complementation between protein surface is in document In coordinated with key, button enters button, raised and groove, donor and acceptor etc. have carried out extensive description, these imply that two phases The property of structure and chemistry matching between interaction surface.In one embodiment, at least one of heterodimer Comprising one or more mutation, wherein the mutation of heavy chain immunoglobulin and light chain immunoglobulin is introduced, in the light of interface New hydrogen bond is introduced between chain and heavy chain.In one embodiment, at least one of heterodimer includes one or many Individual mutation, wherein the mutation of heavy chain immunoglobulin and light chain immunoglobulin is introduced, between the light chain and heavy chain of interface Introduce new salt bridge.

The non-limiting examples of suitable LCCA designs collection are described in embodiment, form and accompanying drawing.In a reality In applying scheme, LCCA designs collection is tied comprising in CH1 domains with least one amino acid modified heavy chain immunoglobulin, CL Have at least one amino acid modified the first light chain immunoglobulin and CL domains in structure domain and repaiied without any amino acid Second light chain immunoglobulin of decorations.In another embodiment, LCCA designs collection is included and have in CH1 domains at least one There is at least one the first amino acid modified immunoglobulin (Ig) in individual amino acid modified heavy chain immunoglobulin, CL domains There is at least one the second amino acid modified light chain immunoglobulin in light chain and CL domains.In another embodiment In, LCCA design collection has at least one amino acid modified heavy chain immunoglobulin, CL domains comprising in CH1 domains Repair with having at least two amino acid at least two amino acid modified the first light chain immunoglobulins and CL domains Second light chain immunoglobulin of decorations.In another embodiment, LCCA designs collection is included and have in CH1 domains at least one There are at least two the first amino acid modified immunoglobulin (Ig)s in individual amino acid modified heavy chain immunoglobulin, CL domains There is at least one the second amino acid modified light chain immunoglobulin in light chain and CL domains.

In one embodiment, LCCA designs collection is included in CH1 domains without amino acid modified immunoglobulin (Ig) weight In chain, CL domains without in amino acid modified the first light chain immunoglobulin and CL domains have at least one amino acid Second light chain immunoglobulin of modification.In one embodiment, LCCA designs collection is included and repaiied without amino acid in CH1 domains Have without in amino acid modified the first light chain immunoglobulin and CL domains in heavy chain immunoglobulin, the CL domains of decorations There are at least two the second amino acid modified light chain immunoglobulins.

In one embodiment, LCCA designs collection is comprising having in CH1 domains at least two amino acid modified to exempt from Have at least without in amino acid modified the first light chain immunoglobulin and CL domains in epidemic disease immunoglobulin heavy chain, CL domains One the second amino acid modified light chain immunoglobulin.In one embodiment, LCCA designs collection is comprising in CH1 domains With at least two amino acid modified heavy chain immunoglobulins, CL domains have at least one amino acid modified first There is at least one the second amino acid modified light chain immunoglobulin in light chain immunoglobulin and CL domains.At one In embodiment, LCCA design collection is included and have in CH1 domains at least two amino acid modified heavy chain immunoglobulins, CL Have in domain at least one amino acid modified the first light chain immunoglobulin and CL domains and have at least two The second amino acid modified light chain immunoglobulin.In one embodiment, LCCA designs collection is included and had in CH1 domains Have at least two amino acid modified heavy chain immunoglobulins, CL domains at least two amino acid modified first immune There are at least two the second amino acid modified light chain immunoglobulins in immunoglobulin light chains and CL domains.In an enforcement In scheme, LCCA design collection is included and have in CH1 domains at least two amino acid modified heavy chain immunoglobulins, CL structures Have in domain at least three amino acid modified the first light chain immunoglobulins and CL domains and there are at least two amino Second light chain immunoglobulin of acid modification.

In one embodiment, LCCA designs collection is comprising having in CH1 domains at least three amino acid modified to exempt from Have at least without in amino acid modified the first light chain immunoglobulin and CL domains in epidemic disease immunoglobulin heavy chain, CL domains One the second amino acid modified light chain immunoglobulin.In one embodiment, LCCA designs collection is comprising in CH1 domains With at least three amino acid modified heavy chain immunoglobulins, CL domains have at least one amino acid modified first There is at least one the second amino acid modified light chain immunoglobulin in light chain immunoglobulin and CL domains.At one In embodiment, LCCA design collection is included and have in CH1 domains at least three amino acid modified heavy chain immunoglobulins, CL Have in domain at least three amino acid modified the first light chain immunoglobulins and CL domains and have at least two The second amino acid modified light chain immunoglobulin.In one embodiment, LCCA designs collection is included and had in CH1 domains Have at least three amino acid modified heavy chain immunoglobulins, CL domains at least four amino acid modified first immune There are at least three the second amino acid modified light chain immunoglobulins in immunoglobulin light chains and CL domains.In an enforcement In scheme, the preferential pairing that LCCA designs are set shown in is by the VH domains of modified immunoglobulin heavy chain, the first immune ball One or more at least one of the VL domains of protein light chain and the VL domains of the second light chain immunoglobulin Amino acid is setting up.The non-limiting examples of suitable LCCA designs collection are as shown in following table lattice and embodiment.

In one embodiment, LCCA designs collection is included in VH domains without amino acid modified immunoglobulin (Ig) weight In chain, VL domains without in amino acid modified the first light chain immunoglobulin and VL domains have at least one amino acid Second light chain immunoglobulin of modification.In one embodiment, LCCA designs collection is included and repaiied without amino acid in VH domains Have without in amino acid modified the first light chain immunoglobulin and VL domains in heavy chain immunoglobulin, the VL domains of decorations There are at least two the second amino acid modified light chain immunoglobulins.

In one embodiment, LCCA designs collection is included and have in VH domains at least one amino acid modified immunity In immunoglobulin heavy chain, VL domains without in amino acid modified the first light chain immunoglobulin and VL domains have at least one Individual the second amino acid modified light chain immunoglobulin.In one embodiment, LCCA designs collection has comprising in VH domains Have at least one amino acid modified heavy chain immunoglobulin, VL domains and have at least one amino acid modified first to exempt from There is at least one the second amino acid modified light chain immunoglobulin in epidemic disease immunoglobulin light chains and VL domains.In a reality In applying scheme, LCCA designs collection is tied comprising in VH domains with least one amino acid modified heavy chain immunoglobulin, VL Have in structure domain at least two amino acid modified the first light chain immunoglobulins and VL domains and there are at least two ammonia Second light chain immunoglobulin of base acid modification.

In one embodiment, LCCA designs collection is included and have in VH domains at least two amino acid modified immunity In immunoglobulin heavy chain, VL domains without in amino acid modified the first light chain immunoglobulin and VL domains have at least one Individual the second amino acid modified light chain immunoglobulin.In one embodiment, LCCA designs collection has comprising in VH domains Have at least two amino acid modified heavy chain immunoglobulins, VL domains and have at least two amino acid modified first to exempt from There is at least one the second amino acid modified light chain immunoglobulin in epidemic disease immunoglobulin light chains and VL domains.In a reality In applying scheme, LCCA designs collection is tied comprising in VH domains with least two amino acid modified heavy chain immunoglobulins, VL Have in structure domain at least one amino acid modified the first light chain immunoglobulin and VL domains and there is at least one ammonia Second light chain immunoglobulin of base acid modification.

In one embodiment, LCCA design collection can be combined to provide comprising two different heavy chain immunoglobulins The combination of (H1 and H2) different with two light chain immunoglobulins (L1 and L2), wherein when H1, H2, L1 and L2 are co-expressed, H1 is preferentially matched with L1, and H2 is preferentially matched with L2.In some embodiments, it is as herein described amino acid modified in double In the linguistic context of specific antibody construct.For example, design collection as herein described can be incorporated into full-length immunoglobulin heavy chain so that The total length heavy chain is preferentially matched with light chain immunoglobulin.In some embodiments, full-length immunoglobulin heavy chain is included Contribute to the amino acid modified of in Fc areas dimerization, as described embodiments.

Specific amino acids modification as herein described is transferred to the transferability of other antibody：

Although the specific amino acids modification of above-mentioned heavy chain immunoglobulin and light chain combines the extracellular knot of D3H44 anti-tissue factors Structure domain antibodies trastuzumab and Cetuximab heavy chain immunoglobulin and light chain are described, but for hereafter, herein Envision and illustrate (referring to embodiment, accompanying drawing and form) these amino acid modified other heavy chain immunoglobulins and light of being transferred to Chain, produces the similar fashion that one of a heavy chain immunoglobulin and two light chain immunoglobulins are preferentially matched.

VH: VL and CH1: CL interface residue is relatively more conservative in interface between heavy chain immunoglobulin and light chain (Padlan etc., 1986, Mol.Immunol.23 (9)：951-960).The sequence conservation is the result for constraining of evolving, and be increased The possibility of the antibody binding domain of functional activation is formed during the combination pairing of light chain and heavy chain.Due to the sequence preservative Property, it is allowed to the sequence modification (it drives preferential pairing) in above-mentioned D3H44 particular instances is transferred to other heavy chains and light chain to different Source dimer, the result being about equal to for preferential pairing, because the area shows that high degree of sequence is guarded in antibody Property；In addition, when occurrence sequence difference, these differences are usually located at the distally at CH1: CL interface.Especially for CH1 and CL knots For structure domain, so situation is exactly.However, with regard to CDR (complementary determining region) ring residue (and length), for particularly CDR-H3, There are some sequence fluctuations at antigen binding site.Therefore, in one embodiment, heterodimer as herein described is to bag Containing such heterodimer：Wherein when antigen binding site amino acid sequence and D3H44 antibody it is dramatically different when, at least One heterodimer is amino acid modified comprising one or more in VH the and/or VL domains positioned at the distally of CDR rings. In another embodiment, heterodimer as herein described is to comprising such heterodimer：Wherein when antigen binding position When the amino acid sequence of point is substantially the same with D3H44 antibody, at least one heterodimer includes the nearside positioned at CDR rings Or one or more in VH the and/or VL domains in distally are amino acid modified.

In one embodiment, it is as herein described amino acid modified antibody to be transferred to according to people or humanization IgG1/ κ Heavy chain immunoglobulin and light chain.The non-limiting examples of such IgG1/ κ chains include difficult to understand (for people) or bent appropriate Monoclonal antibody, handkerchief trastuzumab or bevacizumab (for humanization).

In another embodiment, it is as herein described it is amino acid modified be transferred to using conventional VH and VL subgroups it is anti- The heavy chain immunoglobulin and light chain of body.The non-limiting examples of this antibody-like include handkerchief trastuzumab.

In one embodiment, the amino acid modified antibody for being transferred to the close system genitale of framework as herein described is exempted from Epidemic disease immunoglobulin heavy chain and light chain.The example of this antibody-like includes slave's pearl monoclonal antibody (Obinutuzumab) difficult to understand.

In one embodiment, it is as herein described amino acid modified to be transferred to corner connection between VH: VL domain and closely seen The heavy chain examined and the heavy chain immunoglobulin and light chain of the antibody of the mean value of light chain pair.The example of the type antibody include but not It is limited to handkerchief trastuzumab.In another embodiment, amino acid modified being transferred to as herein described has typical case CL and CH1 The heavy chain immunoglobulin and light chain of the antibody of domain.The suitable example of this antibody-like includes but is not limited to trastuzumab.

In some embodiments, some amino acid modified subsets as herein described are used for antigen binding provided above Variable domains in construct.

Embodiment, accompanying drawing and form show, amino acid modified (for example, at one or many comprising variable region and constant region In individual Fab fragments) other heavy chain immunoglobulins and light chain are transferred to, produce a heavy chain immunoglobulin and two immunity The similar fashion that one of immunoglobulin light chains are preferentially matched.

Preferential pairing

As described above, at least one heterodimer of antigen-binding constructs/heterodimer pair as herein described can One or more comprising its heavy chain immunoglobulin and/or light chain immunoglobulin are amino acid modified so that one heterologous two The heavy chain of aggressiveness such as H1 is preferentially matched with a light chain such as L1 rather than another light chain L2, another heterodimer Heavy chain H2 is preferentially matched with light chain L2 rather than light chain L1.In other words, required preferential pairing is considered as between H1 and L1 And between H2 and L2.Relative to corresponding H1/L1 pair with without one or more each autogamys of amino acid modified H2/L2 pair It is right, when H1 is combined with L1 and L2, if the yield of H1-L1 heterodimers is more than the product of the H1-L2 heterodimers of mispairing Rate, then it is assumed that preferential pairing occurs between such as H1 and L1.Equally, relative to corresponding H1-L1 pair and without one or many The respective pairing of individual amino acid modified H2-L2 pair, when H2 is combined with L1 and L2, if the yield of H2-L2 heterodimers More than the yield of the H2-L1 heterodimers of mispairing, then it is assumed that preferential pairing occurs between H2 and L2.In the linguistic context, bag Heterodimer containing H1 and L1 (H1-L1) or H2 and L2 (H2-L2) be referred to herein as it is preferential pairing, correct pairing, Obligate pairing or required heterodimer, and the heterodimer for including H1 and L2 (H1-L2) or H2 and L1 (H2-L1) exists The referred to herein as heterodimer of mispairing.Two heavy chains and the corresponding catastrophe set of two light chains mean to realize H1-L1 and H2- The selective matching of L2, the catastrophe set referred to as designs collection.

Therefore, in one embodiment, when a heavy chain immunoglobulin and two immune globulins of heterodimer When white light chain is co-expressed, the relative productivity of required heterodimer is more than 55%.In another embodiment, when heterologous two When one heavy chain immunoglobulin of aggressiveness and two light chain immunoglobulins are co-expressed, the relative product of required heterodimer Rate is more than 60%.In another embodiment, when a heavy chain immunoglobulin and two immune globulins of heterodimer When white light chain is co-expressed, the relative productivity of required heterodimer is more than 70%.In another embodiment, when heterologous two When one heavy chain immunoglobulin of aggressiveness and two light chain immunoglobulins are co-expressed, the relative product of required heterodimer Rate is more than 80%.In another embodiment, when a heavy chain immunoglobulin and two immune globulins of heterodimer When white light chain is co-expressed, the relative productivity of required heterodimer is more than 90%.In another embodiment, when heterologous two When one heavy chain immunoglobulin of aggressiveness and two light chain immunoglobulins are co-expressed, the relative product of required heterodimer Rate is more than 95%.

In the above-described example, when H1 and L1 and L2 is co-expressed, if the amount of required H1-L1 heterodimers is more than mistake The amount of the H1-L2 heterodimers matched somebody with somebody, then it is assumed that preferential pairing occurs between H1-L1.Similarly, when the common tables of H2 and L1 and L2 Up to when, if the amount of required H2-L2 heterodimers more than mispairing H2-L2 heterodimers amount, then it is assumed that in H2-L2 Between there is preferential pairing.Therefore, in one embodiment, when heterodimer a heavy chain immunoglobulin and two When light chain immunoglobulin is co-expressed, required heterodimer and the ratio of the heterodimer of mispairing is more than 1.25: 1. In one embodiment, when a heavy chain immunoglobulin and two light chain immunoglobulins of heterodimer are co-expressed, Required heterodimer and the ratio of the heterodimer of mispairing is more than 1.5: 1.In another embodiment, when heterologous two When one heavy chain immunoglobulin of aggressiveness and two light chain immunoglobulins are co-expressed, required heterodimer and mispairing The ratio of heterodimer is more than 2: 1.In another embodiment, when heterodimer a heavy chain immunoglobulin with When two light chain immunoglobulins are co-expressed, required heterodimer and the ratio of the heterodimer of mispairing is more than 3: 1. In another embodiment, when a heavy chain immunoglobulin and two light chain immunoglobulin coexpressions of heterodimer When, required heterodimer and the ratio of the heterodimer of mispairing is more than 5: 1.In another embodiment, when heterologous When a dimeric heavy chain immunoglobulin and two light chain immunoglobulins are co-expressed, required heterodimer and mispairing Heterodimer ratio be more than 10: 1.In another embodiment, when an immunoglobulin (Ig) weight of heterodimer When chain and two light chain immunoglobulins are co-expressed, required heterodimer and the ratio of the heterodimer of mispairing is more than 25 ∶1.In another embodiment, when a heavy chain immunoglobulin and two light chain immunoglobulins of heterodimer are total to During expression, required heterodimer and the ratio of the heterodimer of mispairing is more than 50: 1.

In some embodiments, heterodimer as herein described preferentially matches to form bispecific antibody.One In a little embodiments, the construct comprising preferentially pairing formed selected from D3H44/ trastuzumabs, D3H44/ Cetuximabs and The heterodimer of the bispecific antibody of trastuzumab/Cetuximab.In some embodiments, bispecific antibody bag It is amino acid modified described in 28a-28c containing table.

In some embodiments, two total length heavy chain constructs and the light chain construct coexpression of two uniquenesses, obtain Ten kinds of possible antibody types：H1-L1∶H1-L1、H1-L2∶H1-L2、H1-L1∶H1-L2、H2-L1∶H2-L1、H2-L2∶H2- L2, H2-L1: H2-L2, H1-L1: H2-L1, H1-L2: H2-L2, H1-L2: H2-L1 and H1-L1: H2-L2.H1-L1: H2-L2 kind Class is considered the bispecific antibody species of correct pairing.In some embodiments, select DNA ratios the maximum amount of to obtain The bispecific antibody species of correct pairing.For example, in some embodiments, H1: H2: L1: L2 ratio is 15: 15: 53: 17.In some embodiments, H1: H2: L1: L2 ratio is 15: 15: 17: 53.

In some embodiments, the bispecific species of correct pairing is at least relative to the percentage of all kinds 40%th, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%th, 96%, 97%, 98%, 99% or 100% (see, for example, table 29a-29c and 30a-30c).In some embodiments In, the percentage of the bispecific species of correct pairing is more than corresponding without amino acid modified described in table 28a-28c The percentage of the bispecific species of the correct pairing that wild type H1, H2, L1 and L2 coexpression is obtained.In some embodiments In, with the correct pairing obtained without amino acid modified wild type H1, H2 described in table 28a-28c, L1 and L2 coexpression The percentage of bispecific species is compared, the percentage of the bispecific species of correct pairing reduces at least 5%, 10%, 15%, 20%th, 30%, 40%, 50%, 60%, 70% or 75%.

The heat endurance of heterodimer

In addition to preferential pairing is contributed to, amino acid replacement is selected so that mutation becomes will not Fab heterodimers It is unstable.Therefore, in most of the cases, closely wild type Fab is (for example, for the stability measurement value of Fab heterodimers In 3 DEG C of wild type Fab).

Therefore, in some embodiments, the heat that each heterodimer of heterodimer pair as herein described has Stability is equivalent to comprising identical heavy chain immunoglobulin and light chain but without CH1, CL, VH or VL structure as herein described The amino acid modified heterodimer in domain.In one embodiment, heat endurance is by melt temperature or the measure of Tm.Cause This, in one embodiment, the heat endurance of heterodimer described herein is comprising identical heavy chain immunoglobulin In light chain, about 10 DEG C of the amino acid modified heterodimer without CH1, CL, VH or VL domain as herein described.Cause This, in one embodiment, the heat endurance of heterodimer described herein is comprising identical heavy chain immunoglobulin In light chain, about 5 DEG C of the amino acid modified heterodimer without CH1, CL, VH or VL domain as herein described. In another embodiment, the heat endurance of heterodimer described herein is comprising identical heavy chain immunoglobulin and gently Chain, in about 3 DEG C of the amino acid modified heterodimer without CH1, CL, VH or VL domain as herein described.Another In individual embodiment, the heat endurance of heterodimer described herein comprising identical heavy chain immunoglobulin and light chain, In about 2 DEG C of amino acid modified heterodimer without CH1, CL, VH or VL domain as herein described.In another reality In applying scheme, the heat endurance of heterodimer described herein is including identical heavy chain immunoglobulin and light chain, is not containing In about 1.5 DEG C of the amino acid modified heterodimer of CH1, CL, VH or VL domain as herein described.In another enforcement In scheme, the heat endurance of heterodimer described herein is comprising identical heavy chain immunoglobulin and light chain, without this In about 1 DEG C of the amino acid modified heterodimer of CH1, CL, the VH or VL domain described in text.In another embodiment In, the heat endurance of heterodimer described herein is comprising identical heavy chain immunoglobulin and light chain, without this paper institutes In about 0.5 DEG C of the amino acid modified heterodimer of CH1, CL, the VH or VL domain stated.In another embodiment In, the heat endurance of heterodimer described herein is comprising identical heavy chain immunoglobulin and light chain, without this paper institutes In about 0.25 DEG C of the amino acid modified heterodimer of CH1, CL, the VH or VL domain stated.

Additionally, in some embodiments, the heat endurance of heterodimer described herein is relative to comprising identical Heavy chain immunoglobulin and light chain, the amino acid modified heterodimeric without CH1, CL, VH or VL domain as herein described Body unexpectedly improves (that is, increase).Therefore, in one embodiment, heterodimer described herein is thermally-stabilised Property with comprising identical heavy chain immunoglobulin and light chain, the amino acid without CH1, CL, VH or VL domain as herein described The heterodimer of modification compare increase about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2, 1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、3.0、3.1、3.2、3.3、3.4、3.5、 3.6th, 3.7,3.8,3.9,4.0,4.5,5.0 DEG C or more.

Affinity of the heterodimer to antigen

In one embodiment, each heterodimer of heterodimer pair is identical to the affinity of its respective antigen Or equivalent to comprising identical heavy chain immunoglobulin and light chain but without CH1, CL, VH or VL domain as herein described Amino acid modified heterodimer.In one embodiment, the heterodimer of heterodimer pair is to its respective antigen Affinity comprising identical heavy chain immunoglobulin and light chain, without CH1, CL, VH or VL domain as herein described In about 50 times of amino acid modified heterodimer.In one embodiment, the heterodimer pair of heterodimer pair Its each antigen affinity comprising identical heavy chain immunoglobulin and light chain, without CH1, CL, VH as herein described or In about 25 times of the amino acid modified heterodimer of VL domains.In one embodiment, heterodimer pair is different Source dimer to its each antigen affinity comprising identical heavy chain immunoglobulin and light chain, without as herein described In about 10 times of the amino acid modified heterodimer of CH1, CL, VH or VL domain.In another embodiment, it is heterologous The heterodimer of dimer pair to its each antigen affinity comprising identical heavy chain immunoglobulin and light chain, do not contain In about 5 times of the amino acid modified heterodimer of CH1, CL, VH or VL domain as herein described.In another embodiment party In case, the heterodimer of heterodimer pair to the affinity of its respective antigen comprising identical heavy chain immunoglobulin and Light chain, in about 2.5 times of the amino acid modified heterodimer without CH1, CL, VH or VL domain as herein described. In another embodiment, the heterodimer of heterodimer pair is to the affinity of its respective antigen comprising identical immunity Immunoglobulin heavy chain and light chain, the amino acid modified heterodimer without CH1, CL, VH or VL domain as herein described In about 2 times.In another embodiment, the heterodimer of heterodimer pair is being included to the affinity of its respective antigen It is identical heavy chain immunoglobulin and light chain, amino acid modified different without CH1, CL, VH or VL domain as herein described In source is dimeric about 1.5 times.In another embodiment, the heterodimer of heterodimer pair is to its respective antigen Affinity with comprising identical heavy chain immunoglobulin and light chain, the ammonia without CH1, CL, VH or VL domain as herein described The heterodimer of base acid modification is about the same.

Other optional modification

In one embodiment, the heavy chain immunoglobulin and light chain of heterodimer pair as herein described can be further Modification is (that is, by the covalent attachment of all kinds molecule), so that being covalently attached preferential between without prejudice to heavy chain and light chain Pairing, or affect heterodimer to combine the ability of its antigen, or affect its stability.Such modification is included, but not limited to, e.g. Glycosylation, acetylation, Pegylation, phosphorylation, amidatioon, by known protection/end-capping group derivatization, proteolytic cleavage Cut, be connected to cell ligand or other protein etc..Any various chemical modifications can be carried out by known technology, including but not It is limited to specific chemical cutting, acetylation, formylated, the metabolism of tunicamycin synthesis etc..

In another embodiment, the heavy chain immunoglobulin and light chain of heterodimer pair as herein described can be conjugated (direct or indirect) is to the therapeutic agent or drug moiety for modifying given biological answer-reply.Therapeutic agent or drug moiety should not be explained To be limited to the chemotherapeutics of classics.For example, drug moiety can be the protein with required BA or polypeptide.It is such Protein may include such as anti-knurl enzyme (Onconase) of toxin such as abrin, ricin A, the leopard frog (or another kind of born of the same parents Malicious RNase), PE, cholera toxin or diphtheria toxin；Protein such as TNF, alpha-interferon, β- Interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, apoptosis agent For example TNF-α, TNF-β, AIM I (referring to International Patent Publication No.WO 97/33899), AIM II are (referring to International Patent Publication No.WO 97/34911), FasL (Takahashi etc., 1994, J.Immunol., 6：1567) with VEGI (referring to international special The open No.WO 99/23105 of profit)；Thrombus generating agent or antithrombotic generating agent such as angiostatin or Endostatin；Or biology Answer-reply regulator such as lymphokine (for example, il-1 (" IL-1 "), proleulzin (" IL-2 "), interleukin-6 (" IL-6 "), granulocyte-macrophage colony stimutaing factor (" GM-CSF ") and granulocyte colony stimulating factor (" G-CSF ")) Or growth factor (for example, growth hormone (" GH ")).

Additionally, in an alternate embodiment, antibody can be conjugated to treatment part, such as being conjugated radioactive metal ion Radioactive substance or macrocyclic chelants (seeing above the example of radioactive substance).In certain embodiments, macrocyclic chelate Agent is Isosorbide-5-Nitrae, 7,10- tetraazacyclododecanand-N, N ', N ", "-tetraacethyl (DOTA), it can be connected to anti-N by linkers Body.Such linkers are well known in the art, and in Denardo etc., 1998, Clin Cancer Res.4：2483； Peterson etc., 1999, Bioconjug.Chem.10：553；And Zimmerman etc., 1999, Nucl.Med.Biol.26： It is described in 943.

In some embodiments, the heavy chain immunoglobulin and light chain of heterodimer is with the fusion protein comprising label Expression, the label is conducive to purifying and/or tests.As described herein, " label " is the amino acid series of any addition, can be deposited It is C- ends, N- ends or the inside of protein, to contribute to identification or the purifying of protein.Suitable label include but It is not limited to the label for purifying and/or testing well known by persons skilled in the art, such as albumin binding domain (ABD), His labels, FLAG labels, glutathione-s- transferases, hemagglutinin (HA) and maltose-binding protein.Such labelled protein Can engineered be comprising cleavage site, such as fibrin ferment, enterokinase or factor X cleavage site, in order to before purification, the phase Between or remove label afterwards.

In some embodiments, light chain (position 214, Kabat numberings) and the heavy chain (position of interchain disulfide bond are formed 233, Kabat numbering) Fab domains bottom one or more cysteine residues can be modified to serine or alanine or Non- cysteine or different amino acid.

Imagination heavy chain immunoglobulin can be made it is other amino acid modified, to improve preferential pairing level and/or different The heat endurance of source dimer pair.For example, heavy chain immunoglobulin Fc domains can be made it is other amino acid modified, to drive Dynamic heterodimer is to relative to the preferential pairing between homodimer pair.It is such it is amino acid modified be it is known in the art, And including such as United States Patent (USP), those modifications of No.2012/0149876 descriptions are disclosed.Or, it is possible with driving heterologous two Aggressiveness to relative to the alternative strategy of the preferential pairing between homodimer pair, such as, " button enters button ", with ion phase The charged residues of interaction and chain exchange engineered domain (SEED) technology.Strategy below has been retouched in the art State, and in Klein etc., commented during ibid.Fc domains are further discussed below.

Fc domains

Wherein in embodiment of the antigen-binding polypeptides construct comprising full-length immunoglobulin heavy chain, construct will be wrapped Containing Fc.In some respects, the Fc includes at least one or two CH3 domain sequences.In some respects, antigen-binding polypeptides are worked as When construct includes heterodimer (the Fab areas only comprising heavy chain), the Fc by or do not connected by one or more joints To the first heterodimer and/or the second heterodimer.In some respects, the Fc is people Fc.In some respects, the Fc is people IgG or IgG1Fc.In some respects, the Fc is heterodimer Fc.In some respects, the Fc includes at least one or two CH2 domain sequences.

In some respects, the Fc includes C_H3One or more modifications at least one of domain sequence.At some Aspect, the Fc includes C_H2One or more modifications at least one of domain sequence.In some respects, Fc is that wall scroll is more Peptide.In some respects, Fc is a plurality of peptide, such as two polypeptides.

In some respects, the Fc includes C_H3The one or more modifications of at least one of sequence.In some respects, the Fc Comprising C_H2One or more modifications at least one of sequence.In some respects, Fc is wall scroll polypeptide.In some respects, Fc is a plurality of peptide, such as two polypeptides.

In some respects, Fc is to be filed in the patent application PCT/CA2011/001238 on November 4th, 2011 or be filed in Fc described in the PCT/CA2012/050780 on November 2nd, 2012, the complete disclosure of each patent application is accordingly drawing Mode is integrally incorporated for all purposes.

In some respects, construct as herein described includes such heterodimer Fc：It includes the asymmetric modifications of Jing Modification CH3 domains.Heterodimer Fc can include two heavy chain constant domain polypeptides：First heavy chain polypeptide and the second weight Chain polypeptide, they are used interchangeably, and precondition is that Fc includes first heavy chain polypeptide and second heavy chain polypeptide.Typically For, the first heavy chain polypeptide includes a CH3 sequences, and the second heavy chain polypeptide includes the 2nd CH3 sequences.

When two CH3 sequence dimerizations, comprising introducing one or more amino acid modified two in an asymmetrical fashion CH3 sequences generally produce heterodimer Fc rather than homodimer.As used herein, " asymmetric amino acid modification " refer to it In the amino acid of specific location in a CH3 sequences be different from appointing for amino acid at same position in the 2nd CH3 sequences What is modified, and the first and second CH3 sequences preferentially match to form heterodimer rather than homodimer.This heterodimeric Change can only modify in each sequence at identical orresponding amino acid position one of two amino acid；Or the first He of modification The result of two amino acid in each sequence of the identical corresponding position of each in the 2nd CH3 sequences.Heterodimer The first and second CH3 sequences of Fc can be modified comprising one or more than an asymmetric amino acid.

Table X provides the amino acid sequence of the corresponding human IgG1 Fc sequences of amino acid 231 to 447 of total length human IgG1's heavy chain Row.Amino acid 341-447 of the CH3 sequences comprising total length human IgG1's heavy chain.

Generally Fc may include two that are capable of dimerization adjacent sequence of heavy chain (A and B).In some respects, of Fc Or two sequences are including one or more mutation at following position or modify：L351、F405、Y407、T366、K392、T394、 T350, S400 and/or N390 (are numbered) using EU.In some respects, Fc includes the mutant sequence shown in Table X.In some sides Face, Fc includes the mutation of variant 1A-B.In some respects, Fc includes the mutation of variant 2A-B.In some respects, Fc includes variant The mutation of 3A-B.In some respects, Fc includes the mutation of variant 4A-B.In some respects, Fc includes the mutation of variant 5A-B.

First and second CH3 sequences can include the ammonia described in the amino acid 231 to 447 herein in conjunction with total length human IgG1's heavy chain Base acid mutation.In one embodiment, heterodimer Fc includes modified CH3 domains, wherein CH3 sequences tool Have amino acid modified at position F405 and Y407, and the 2nd CH3 sequences have it is amino acid modified at the T394 of position.One In individual embodiment, heterodimer Fc include modified CH3 domains, wherein a CH3 sequences have selected from L351Y, One or more of F405A and Y407V are amino acid modified, and the 2nd CH3 sequences have selected from T366L, T366I, K392L, K392M It is amino acid modified with one or more of T394W.

In one embodiment, heterodimer Fc includes modified CH3 domains, wherein CH3 sequences tool Have amino acid modified at position L351, F405 and Y407, the 2nd CH3 sequences have the ammonia at position T366, K392 and T394 Base acid modification, first or the 2nd one of CH3 sequences also comprising amino acid modified at the Q347 of position, other CH3 sequences are also Comprising amino acid modified at the K360 of position.In another embodiment, heterodimer Fc includes modified CH3 structures Domain, wherein a CH3 sequences have amino acid modified at position L351, F405 and Y407, the 2nd CH3 sequences have position It is amino acid modified at T366, K392 and T394, first or the 2nd one of CH3 sequences also comprising the amino at the Q347 of position Acid modification, other CH3 sequences also comprising amino acid modified at the K360 of position, also wrap by one of described CH3 sequences or both Containing amino acid modified T350V.

In one embodiment, heterodimer Fc includes modified CH3 domains, wherein CH3 sequences tool Have amino acid modified at position L351, F405 and Y407, the 2nd CH3 sequences have the ammonia at position T366, K392 and T394 One of base acid modification, described first and second CH3 sequences also include amino acid modified D399R or D399K, other CH3 sequences Row include one or more in T411E, T411D, K409E, K409D, K392E and K392D.In another embodiment, Heterodimer Fc includes modified CH3 domains, wherein a CH3 sequences have at position L351, F405 and Y407 Amino acid modified, the 2nd CH3 sequences have amino acid modified at position T366, K392 and T394, first and second CH3 One of sequence also include amino acid modified D399R or D399K, other CH3 sequences comprising T411E, T411D, K409E, One of one or more in K409D, K392E and K392D, described CH3 sequences or the two also comprising amino acid modified T350V。

In one embodiment, heterodimer Fc includes modified CH3 domains, wherein CH3 sequences tool Have amino acid modified at position L351, F405 and Y407, the 2nd CH3 sequences have the ammonia at position T366, K392 and T394 Base acid modification, wherein one of described CH3 sequences or both also include amino acid modified T350V.

In one embodiment, heterodimer Fc is included comprising following amino acid modified modified CH3 structures Domain, wherein " A " represents the amino acid modified of a CH3 sequences, " B " represents the amino acid modified of the 2nd CH3 sequences：A：L351Y_ F405A_Y407V、B：T366L_K392M_T394W、A：L351Y_F405A_Y407V、B：T366L_K392L_T394W、A： T350V_L351Y_F405A_Y407V、B：T350V_T366L_K392L_T394W、A：T350V_L351Y_F405A_Y407V、 B：T350V_T366L_K392M_T394W、A：T350V_L351Y_S400E_F405A_Y407V and/or B：T350V_T366L_ N390R_K392M_T394W。

One or more asymmetric amino acid modifications can help to the formation of heterodimer Fc, wherein heterodimer CH3 domains have the stability equivalent to wild-type homologous dimer CH3 domains.In one embodiment, one or Multiple asymmetric amino acid modifications contribute to the formation of heterodimer Fc domains, and wherein heterodimer Fc domains have Equivalent to the stability of wild-type homologous dimeric Fc domain.In one embodiment, one or more asymmetric amino Acid modification contributes to the formation of heterodimer Fc domains, and wherein heterodimer Fc domains have in means of differential scanning calorimetry By the stability of melt temperature (Tm) observation in research, and wherein melt temperature is in observed corresponding symmetrical wild type In 4 DEG C of homodimer Fc domains.In some respects, Fc includes C_H3One or more at least one of sequence are repaiied Decorations, the modification contributes to forming heterodimer Fc of the stability equivalent to wild-type homologous dimeric Fc.

In one embodiment, the stability of CH3 domains can be surveyed by, for example, differential scanning calorimetry (DSC) Determine the melt temperature of CH3 domains to assess.Therefore, in a further embodiment, CH3 domains have about 68 DEG C or higher Melt temperature.In another embodiment, CH3 domains have about 70 DEG C or higher melt temperature.In another reality In applying scheme, CH3 domains have about 72 DEG C or higher melt temperature.In another embodiment, CH3 domains have About 73 DEG C or higher melt temperature.In another embodiment, CH3 domains have about 75 DEG C or higher melting temperature Degree.In another embodiment, CH3 domains have about 78 DEG C or higher melt temperature.In some respects, dimerization C_H3 Sequence has about 68,69,70,71,72,73,74,75,76,77,77.5,78,79,80,81,82,83,84 or 85 DEG C or higher Melt temperature (Tm).

In some embodiments, compared with the homodimer Fc in expression product, comprising modified CH3 sequences Heterodimer Fc can at least about 75% purity formed.In another embodiment, heterodimer Fc is with greater than about 80% purity is formed.In another embodiment, heterodimer Fc is formed with the purity for being greater than about 85%.At another In embodiment, heterodimer Fc is formed with the purity for being greater than about 90%.In another embodiment, heterodimer Fc Formed with the purity for being greater than about 95%.In another embodiment, heterodimer Fc is formed with the purity for being greater than about 97%. In some respects, Fc be in expression to be greater than about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%th, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or The heterodimer that 99% purity is formed.In some respects, Fc be when being expressed by individual cells to be greater than about 75%, 76%th, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%th, the heterodimer that 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity is formed.

Monomer Fc polypeptides are modified to contribute to the other method of heterodimer Fc formation in International Patent Publication (Gunasekaran K.et al. (2010) the J Biol such as No.WO 96/027011 (knob enters button), Gunasekaran Chem.285,19637-46, electrostatic design to achieve selective Heterodimerization), (Davis, JH.et al. (2010) the Prot Eng Des Sel such as Davis；23(4)：195- 202, strand exchange engineered domain (SEED) technology) and [Efficient such as Labrijn generation of stable bispecific IgG1by controlled Fab-arm exchange.Labrijn AF, Meesters JI, de Goeij BE, van den Bremer ET, Neijssen J, van Kampen MD, Strumane K, Verploegen S, Kundu A, Gramer MJ, van Berkel PH, van de Winkel JG, Schuurman J, Parren PW.Proc Natl Acad Sci US A.2013Mar 26；110(13)：5145-50] in have It is described.In some embodiments, detached construct as herein described includes the antigen-binding constructs of conjugated antigen；With And relative to the antigen-binding constructs of identical Fc polypeptide are not included, with excellent biophysical properties such as stability, And easily prepared dimeric Fc polypeptide construct.Many mutation in the sequence of heavy chain of Fc are known in the art optional Sexually revise affinity of the antibody Fc to different Fc γ acceptors.In some respects, the Fc includes the selection for contributing to Fc- γ acceptors Property combine one or more modifications.

CH2 domains are the amino acid 231-340 of the sequence shown in Table X.Exemplary mutations are as follows：

S298A/E333A/K334A, S298A/E333A/K334A/K326A (Lu Y, Verne s JM, Chiang N etc., J Immunol Methods., on 2 28th, 2011,365 (1-2)：132-41)；F243L/R292P/Y300L/V305I/ P396L, F243L/R292P/Y300L/L235V/P396L (Stavenhagen JB, Gorlatov S, Tuaillon N etc., Cancer Res., on September 15th, 2007,67 (18)：8882-90；Nordstrom JL, Gorlatov S, Zhang W etc., Breast Cancer Res., on November 30th, 2011,13 (6)：R123)；F243L(Stewart R、Thom G、Levens M Deng, Protein Eng Des S el., in September, 2011,24 (9)：671-8.)、S298A/E333A/K334A(Shields RL, Namenuk AK, Hong K etc., J Biol Chem., March 2 calendar year 2001,276 (9)：6591-604)；S239D/ I332E/A330L, S239D/I332E (Lazar GA, Dang W, Karki S etc., Proc Natl Acad Sci U S A., On March 14th, 2006,103 (11)：4005-10)；S239D/S267E、S267E/L328F(Chu SY、Vostiar I、Karki S etc., Mol Immunol., in September, 2008,45 (15)：3926-33)；S239D/D265S/S298A/I332E、S239E/ S298A/K326A/A327H、G237F/S298A/A330L/I332E、S239D/I332E/S298A、S239D/K326E/ A330L/I332E/S298A、G236A/S239D/D270L/I332E、S239E/S267E/H268D、L234F/S267E/ Other mutation that N325L, G237F/V266L/S267D and WO2011/120134 and WO2011/120135 are listed, these are dashed forward Change is hereby incorporated herein by.Therapeutic Antib ody Engineering(by William R.Strohl And Lila M.Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 In October, 907568 37 9,2012) page 283 list mutation.

In some embodiments, CH2 domains include one or more asymmetric amino acid modifications.In some embodiment party In case, one or more the asymmetric amino acid modifications of CH2 domains comprising the selective binding for contributing to Fc γ R.At some In embodiment, CH2 domains allow the separation of detached construct as herein described and purifying.

FcRn is combined and PK parameters

As it is known in the art, the combination of FcRn make the antibody of endocytosis from endosome loop back blood flow (Raghavan etc., 1996, Annu Rev Cell Dev Biol 12：181-220；Ghetie etc., 2000, Annu Rev Immunol 18：739- 766).The process is filtered with kidney and excludes (because the molecular weight of full-length molecule is larger) combination, is obtained in one to three all scopes The favourable antibody serum half-life.Fc is attached to FcRn and key effect is also played in antibody delivery.Therefore, in an embodiment In, the construct of the present invention can be with reference to FcRn.

Improve the other modification of effector function.

In some embodiments, construct as herein described can be modified, to improve its effector function.Such modification is It is known in the art, it is fucosylated including going, or engineering reform antibody Fc part to activate acceptor affinity it is main Be FCGR3a to ADCC and C1q to CDC.Following table Y summarize report in document for engineered various of effector function Design.

Therefore, in one embodiment, construct as herein described may include amino acid modified comprising one or more Dimeric Fc, the amino acid modified effector function that improvement is given as described in upper table.In another embodiment, structure Build body can go it is fucosylated, to improve effector function.

Joint

Construct as herein described may include one or more heterodimers as herein described, and the heterodimer can be grasped It is coupled to Fc as herein described with making.In some respects, Fc by or be not coupled to by one or more joints one Or multiple heterodimers.In some respects, Fc is directly coupled to one or more of heterodimers.In some respects, Fc is coupled to one or more of heterodimers by one or more joints.In some respects, Fc is coupled by joint To the heavy chain of each heterodimer.

In some respects, one or more of joints are one or more peptide linkers.In some respects, it is one Or multiple joints include one or more antibody hinge regions.In some respects, one or more of joints include one or many Individual IgG1 hinge areas.

The preparation method of heterodimer pair

As described above, heterodimer pair as herein described can include the first heterodimer and the second heterodimer, Each heterodimer is included has at least heavy chain immunoglobulin of VH and CH1 domains or its fragment, and with VL knots Structure domain and the light chain immunoglobulin of CL domains.The heavy chain immunoglobulin and light chain immunoglobulin of heterodimer can be easy In being prepared using recombinant DNA technology known in the art.Standard technique such as Sambrook and Russell, Molecular Cloning：A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., the 3rd edition, 2001)；Sambrook etc., Molecular Cloning：A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., second edition, 1989)；Short Protocols in Molecular Biology (Ausubel etc., John Wiley and Sons, New York, the 4th edition, 1999)；And Glick and Pasternak, Molecular Biotechnology：Principles (1998) ASM Press, Washington, D.C., second edition describe and Applications of Recombinant DNA Those technologies can be used for recombinant nucleic acid method, nucleic acid synthesis, cell culture, transgenosis mix and recombinant protein expression.Or Person, heterodimer as herein described and heterodimer are to being chemical synthesis.

The heavy chain immunoglobulin of the antibody of derivative heterodimer and the nucleic acid of light chain and amino acid sequence are this areas It is known, or can easily be used nucleic acid and/or protein sequencing methods measure.By label Gene Fusion as herein described to immunity The method of immunoglobulin heavy chain and/or light chain is known in the art, and some in these methods below with embodiment in It is described.

For example, express in host cell and be co-expressed heavy chain immunoglobulin and light chain method be it is well known that 's.Additionally, marking heavy chain and/or the method for light chain to be also well known in using recombinant DNA technology.Suitable for heavy chain and gently The expression vector and host cell of chain expression is also well known in, as mentioned below.

Bispecific antibody preparation method is not relied on only using the monoclonal or transient cell line of whole four chains of expression, The method is (Gramer etc., (2013) mAbs 5,962 known in the art；Strop etc., (2012) J Mol Biol 420, 204.).These methods rely only on the oxygen in the two pairs of light chains and heavy chain for being related to bispecific antibody formation (redox generation) The generation postbrachium that change is carried out under reducing condition is exchanged.In the method, H1: L1 can be in two kinds of different clones with H2: L2 pair Middle expression, to have independently produced two Fab arms.Subsequently, two Fab arms are mixed under the conditions of selective redox, to realize two Bar unique heavy chain H1's and H2 associates again, to form the bispecific antibody comprising L1: H1: H2: L2 chain.It is contemplated that using this Design library/data set described in text is prepared bispecific using the improved form of redox generation method or the method and is resisted Body.

In some embodiments, using cell-free protein expression system, and living cells is not used come the polypeptide (example that is co-expressed Such as, heavy chain and light chain polypeptide).Conversely, DNA being transcribed into RNA and RNA being translated as into all components needed for protein (for example Ribosomes, tRNA, enzyme, co-factor, amino acid) with solution offer, for using in vitro.In certain embodiments, vivoexpression The hereditary template (mRNA or DNA) and (2) of needs (1) encoding heavy chain and light chain polypeptide is comprising necessary transcription and translation molecule The reaction solution of machine.In certain embodiments, cell extract generally provides the component of reaction solution, for example：With RNA polymerase, the ribosomes for polypeptide translation, tRNA, amino acid, enzyme cofactor, energy source and albumen in mRNA transcriptions The critical cellular components that matter is correctly folded.Cell-free protein expression system can be using from bacterial cell, yeast cells, elder brother It is prepared by worm cell, plant cell, mammalian cell, the lysate of people's cell or combinations thereof.Such cell lysate can The correct composition and ratio of the required enzyme of translation and construction unit are provided.In some embodiments, cell membrane is removed, is only stayed The cytosol and organelle component of lower cell.

Many cell-free protein expression systems are known in the art, such as Carlson, (2012) Biotechnol.Adv.30：1185-1194 is commented.For example, cell-free protein expression system can be thin based on protokaryon or eucaryon Born of the same parents obtain.The example of protokaryon Cell free expression system includes those from Escherichia coli (E.coli.).Eucaryon acellular albumen Matter expression system can be based on the extract of such as rabbit desmacyte, wheat embryo and insect cell and obtain.Such protokaryon and eucaryon Cell-free protein expression system can be commercially available from the company of such as Roche, Invitrogen, Qiagen and Novagen.This The technical staff in field can be easy to the suitable nothing for selecting that polypeptide (for example, heavy chain and light chain polypeptide) that can be paired with each other can be generated Cell protein expression system.In addition, cell-free protein expression system can also supplement molecular chaperones (such as BiP) and isomerase (for example, disulfide bond isomerase), to improve the efficiency of IgG foldings.

In some embodiments, using Cell free expression system coexpression from DNA profiling (transcription and translation) or The heavy chain and light chain polypeptide of mRNA templates (only translating).

Carrier and host cell

The recombinant expressed needs of heavy chain and light chain are built comprising encoding heavy chain or light chain (for example, antibody or fusion protein) The expression vector of polynucleotides.Once the polynucleotides of encoding heavy chain or light chain are obtained, for generating the carrier of heavy chain or light chain Can be generated by recombinant DNA technology using technology well known in the art.Therefore, this document describes including encoding heavy chain by expression Or the polynucleotides of the nucleotide sequence of light chain are preparation method of protein.Method well-known to those having ordinary skill in the art can use The expression vector comprising heavy chain or light chain encoding sequences and appropriate transcription and translation control signal in structure.These methods include Such as recombinant DNA technology in vi, synthetic technology and internal Genetic Recombination.Therefore, the present invention is provided to include and may be operably coupled to The replicable vector of the encoding heavy chain of promoter or the nucleotide sequence of light chain.

Expression vector is transferred to by host cell by routine techniques, then by routine techniques culture transfectional cell, with Generate the modified heavy chain or light chain for the method for the present invention.In particular embodiments, the common table in host cell Up to heavy chain and light chain used by the method, to express whole immunoglobulin molecules, as detailed below.

Many host-expression vector systems can be used to express modified heavy chain and light chain.Such host expression system is represented The instrument of coded sequence of interest can be generated and subsequently be purified, is also represented by converting or transfecting appropriate nucleotide coding sequence When, can the modified heavy chain of expression in situ and light chain cell.These host expression systems include but is not limited to microorganism, such as Conversion has the recombinant phage dna comprising modified heavy chain and light chain encoding sequences, DNA or cosmid DNA expression vectors Bacterium (for example, Escherichia coli and bacillus subtilis (B.subtilis))；Conversion has comprising modified heavy chain and light chain Yeast (for example, saccharomyces (Saccharomyces), the pichia of the recombinant yeast expression vector of coded sequence (Pichia))；Transfection has (for example, the shaft-like disease of the recombinant virus expression vector comprising modified heavy chain and light chain encoding sequences Poison) insect cell system；(for example, transfection has comprising modified heavy chain and light chain encoding sequences recombinant virus expression vector Cauliflower mosaic virus, CaMV；Tobacco mosaic virus (TMV), TMV) or conversion have comprising modified heavy chain and light chain encoding sequences weight The plant cell system of group plasmid expression vector (for example, Ti-plasmids)；Or with comprising from mammalian cell gene group Promoter (for example, metallothionein promoter) or from the promoter of mammalian virus, (for example, adenovirus late is opened Mover；Vaccinia virus 7.5K promoters) recombinant expression construct body mammalian cell system (for example, COS, CHO, BHK, HEK-293, NSO and 3T3 cell).In certain embodiments, bacterial cell such as Escherichia coli or eukaryotic are used for into table Up to modified heavy chain and light chain, the heavy chain and light chain are recombinant antibodies or fusion protein molecule.For example, mammalian cell (such as Chinese hamster ovary cell (CHO)), with reference to carrier (such as from the main immediate early gene of people's cell hugeization virus Promoter element) be antibody effective expression system (Foecking etc., 1986, Gene 45：101；With Cockett etc., 1990, Bio/Technology 8：2).In a particular embodiment, the heavy chain immunoglobulin and light chain of each heterodimer are encoded Nucleotide sequence expression by constitutive promoter, inducible promoter or tissue-specific promoter regulate and control.

In mammalian host cell, using many expression systems based on virus.Adenovirus tabulation wherein Up to carrier in the case of, modified heavy chain and light chain encoding sequences of interest may be connected to adenovirus transcription/translation control Compound, such as late promoter and tripartite leader[Ru Jianyuxianbingdu].Then the mosaic gene can insert gland by external or In vivo recombination Viral genome.Inserting virus genomic nonessential region (for example, region E1 or E3) will produce surviving and can feel Express in the host of dye modified heavy chain and light chain recombinant virus (for example, with reference to Logan＆Shenk, 1984, Proc.Natl.Acad.Sci.USA 81：355-359).Specific initial signal can also be the antibody coding sequence of insertion Effectively translate necessary.These signals include ATG initiation codon and adjacent sequence.Additionally, initiation codon must be with institute The reading frame homophase of the coded sequence for needing, to guarantee the translation of whole insetion sequence.These Exogenous translational control signals and starting Codon can have many sources, natural and synthesis.The efficiency of expression can by including appropriate transcription enhancer element, Transcription terminator etc. (see, for example, Bittner etc., 1987, Methods in Enzymol.153 to improve：516-544).

The heavy chain immunoglobulin of heterodimer and the expression of light chain can by any promoter known in the art or Enhancer element is controlled.Can be used to control the gene table of the modified heavy chain of coding and light chain (for example, antibody or fusion protein) The promoter for reaching include but is not limited to SV40 early promoters area (Bernoist and Chambon, 1981, Nature 290： 304-310), include in 3 ' LTRs of Rous sarcoma virus promoter (Yamamoto etc., 1980, Cell 22：787-797), herpes thymidine kinase promoter (Wagner etc., 1981, Proc.Natl.Acad.Sci.U.S.A.78.1441-1445), metallothionein gene regulating and controlling sequence (Brinster etc., 1982, Nature 296：39-42), tetracycline (Tet) promoter (Gossen etc., 1995, Proc.Nat.Acad.Sci.USA 89：5547-5551)；Procaryotic cell expression carrier beta-lactamase promoter (Villa-Kamaroff etc., 1978, Proc.Natl.Acad.Sci.U.S.A.75：3727-3731) or tac promoters (DeBoer etc., 1983, Proc.Natl.Acad.Sci.U.S.A.80：21-25；Referring also to " Useful proteins from recombinant Bacteria " in Scientific American, 1980,242：74-94)；Plant comprising rouge alkali synthetase promoter area Thing expression vector (Herrera-Estrella etc., Nature 303：209-213) or cauliflower mosaic virus 35S RNA start Son (Gardner etc., 1981, Nucl.Acids Res.9：And photosynthesis enzyme carboxydismutase is opened 2871) Mover (Herrera-Estrella etc., 1984, Nature 310：115-120)；The promoter element of yeast or other fungies is all As the promoters of Gal 4, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerokinase) promoter, alkaline phosphatase promoter with And tissue specificity is shown below and for the animal transcriptional control zone of transgenic animals：It is active in pancreatic acinar cell Elastase I gene control zone (Swift etc., 1984, Cell 38：639-646；Ornitz etc., 1986, Cold Spring Harbor Symp.Quant.Biol.50：399-409；MacDonald, 1987, Hepatology 7：425- 515)；In pancreatic beta cell activated insulin gene control region (Hanahan, 1985, Nature 315：115-122)、 In lymphocyte activated immunoglobulin gene control zone (Grosschedl etc., 1984, Cell 38：647-658； Adames etc., 1985, Nature 318：533-538；Alexander etc., 1987, Mol.Cell.Biol.7：1436-1444)、 In testis, mammary gland, lymph and mast cell activated mouse mammary adenoma virus control zone (Leder etc., 1986, Cell 45：485-495), activated albumin gene control region (Pinkert etc., 1987, Genes and in liver Devel.1：268-276), in liver activated α-fetoprotein gene-controlled area (Krumlauf etc., 1985, Mol.Cell.Biol.5：1639-1648；Hammer etc., 1987, Science 235：53-58)；In liver activated α- 1- antitrypsin genes control zone (Kelsey etc., 1987, Genes and Devel.1：161-171), in bone marrow cell Activated betaglobulin gene-controlled area (Mogram etc., 1985, Nature 315：338-340；Kollias etc., 1986, Cell 46：89-94)；Activated MBP gene control zone in oligodendroglia in the brain (Readhead etc., 1987, Cell 48：703-712)；The activated gene-controlled area of myosin light chain -2 in skeletal muscle (Sani, 1985, Nature 314：283-286)；The activated neuronspecific enolase in neuronal cell (NSE) (Morelli etc., 1999, Gen.Virol.80：571-83)；The activated brain source nerve battalion in neuronal cell The foster factor (BDNF) gene-controlled area (Tabuchi etc., 1998, Biochem.Biophysic.Res.Com.253：818-823)； Activated glial fibrillary acidic protein (GFAP) promoter (Gomes etc., 1999, Braz J Med in astrocyte Biol Res 32(5)：619-631；Morelli etc., 1999, Gen.Virol.80：It is 571-83) and active in hypothalamus Gonadotropin releasing hormone gene control zone (Mason etc., 1986, Science 234：1372-1378).

Additionally, the expression of regulation insetion sequence may be selected or with required ad hoc fashion modification and the place of processed gene product Chief cell strain.The expression from some promoters can be improved in the case where there are some inducers；Therefore, can control genetic engineering Fusion protein expression.Additionally, different host cells has for translation and post translational processing and modification (for example, albumen The glycosylation of matter, phosphorylation) feature and specific mechanism.It is expressed to guarantee that appropriate clone or host system may be selected The required modification of exogenous proteins and processing.For example, expression will generate not glycosylated product in bacterial system, in yeast Expression will generate glycosylated product.The appropriate processing that the primary transcript with gene outcome can be used (for example, is glycosylated And phosphorylation) cellular machineries eukaryotic host cell.Such mammalian host cell include but is not limited to CHO, VERY, BHK, Hela, COS, MDCK, HEK-293,3T3, WI38, NSO, especially neuronal cell system, such as SK-N-AS, SK- N-FI, SK-N-DZ HNB (Sugimoto etc., 1984, J.Natl.Cancer Inst.73：51-57)、SK-N- SH HNBs (Biochim.Biophys.Acta, 1982,704：450-460), Daoy people's cerebellum medulloblastoma (He etc., 1992, Cancer Res.52：1144-1148), DBTRG-05MG glioblastoma cells (Kruse etc., 1992, In Vitro Cell.Dev.Biol.28A：609-614), IMR-32 HNBs (Cancer Res., 1970,30： 2110-2118), 1321N1 Human astrocytes knurl (Proc.Natl.Acad.Sci.USA, 1977,74：4816)、MOG-G-CCM Human astrocyte knurl (Br.J.Cancer, 1984,49：269), U87MG people's glioblastoma-astrocytoma (Acta Pathol.Microbiol.Scand., 1968,74：465-486), A172 people's glioblastoma (Olopade etc., 1992, Cancer Res.52：2523-2529), C6 rat Glioma cells (Benda etc., 1968, Science 161：370-371)、 Neuro-2a mouse neuroblastomas (Proc.Natl.Acad.Sci.USA, 1970,65：129-136), NB41A3 mouse god Jing blastomas (Proc.Natl.Acad.Sci.USA, 1962,48：1184-1190), SCP sheep choroid plexus (Bolin etc., 1994, J.Virol.Methods 48：211-221), the normal astrocyte of G355-5, PG-4 cat (Haapala etc., 1985, J.Virol.53：827-833), Mpf ferrets brain (Trowbridge etc., 1982, In Vitro 18：It is 952-960) and normal Clone, such as CTX TNA2 rats Normal brain cortex (Radany etc., 1992, Proc.Natl.Acad.Sci.USA 89：6467-6471), such as CRL7030 and Hs578Bst.Additionally, different carrier/host expression systems can be with difference Degree affect processing reaction.

For the long-term of recombinant protein, high yield are generated, stable expression is typically preferred.For example, can engineering change Make the modified heavy chain of the stable expression present invention and the clone of light chain (for example, antibody or fusion protein).Host cell can The appropriate expression control element (for example, promoter, enhancer, sequence, transcription terminator, site of polyadenylation etc.) of conversion and The DNA of selected marker control, and do not use the expression vector comprising virus origin of replication.After foreign DNA is introduced, it is allowed to work The cell of journey transformation grows 1-2 days in enriched medium, then switches to Selective agar medium.Selectivity mark in recombinant plasmid Note gives and selects resistance, it is allowed to which plasmid stabilisation is integrated into cell into chromosome and growth forms stove, the stove can be cloned then and is expanded Greatly to clone.

Many selection systems can be used, the simple of tk-, hgprt- or aprt- cell can be including but not limited to respectively used to Herpesvirus thymine deoxyriboside kinase (Wigler etc., 1977, Cell 11：223), hypoxanthine-guanine monophosphate nucleotidyltransferase (Szybalska＆Szybalski, 1962, Proc.Natl.Acad.Sci.USA 48：2026) shift with adenine phosphoric acid nucleoside Enzyme (Lowy etc., 1980, Cell 22：817) gene.In addition, antimetabolite resistance can be used as dhfr (imparting methotrexate resistance) (Wigler etc., 1980, Natl.Acad.Sci.USA 77：3567；O ' Hare etc., 1981, Proc.Natl.Acad.Sci.USA 78：1527)；Gpt (imparting mycophenolic acid) (Mulligan＆Berg, 1981, Proc.Natl.Acad.Sci.USA 78： 2072)；Neo (giving aminoglycoside G-418 resistances) (Colberre-Garapin etc., 1981, J.Mol.Biol.150：1)；With Hygro (imparting hygromycin resistance) (Santerre etc., 1984, Gene 30：147) basis of gene selects.

The coexpression of heavy chain and light chain

The heavy chain immunoglobulin and light chain of heterodimer pair as herein described can be co-expressed in mammalian cell, As described above.In one embodiment, a heavy chain light chain different from two is co-expressed with above-mentioned LCCA design collection, its Middle heavy chain preferentially with the pairing of one of two light chains.In another embodiment, the heavy chain of two uniquenesses and two are solely Special light chain coexpression, wherein every heavy chain is preferentially matched with a light chain.

The test of heterodimer pair

As described above, at least one heterodimer of heterodimer pair as herein described may include its immunoglobulin (Ig) One or more of heavy chain and/or light chain immunoglobulin are amino acid modified so that when two uniquenesses of heterodimer pair When the light chain of heavy chain and two uniquenesses is co-expressed in mammalian cell, the heavy chain of the first heterodimer preferentially with one Light chain rather than another pairing.Equally, the heavy chain of the second heterodimer is preferentially matched with the second light chain rather than the first light chain. Can for example using the degree of the preferential pairing of method described below assessment.Heterodimer pair can as mentioned below be tested The affinity of each heterodimer and its respective antigen.Can also test as mentioned below heterodimer pair each heterologous two The heat endurance of aggressiveness.

The assay method of preferential pairing

LCCA

In one embodiment, the preferential pairing between heavy chain immunoglobulin and light chain is by carrying out light chain competition point Analyse (LCCA) to determine.Jointly owned patent application PCT/the US2013/063306 for being filed on October 3rd, 2013 is described The various embodiments of LCCA, the patent application is incorporated herein in its entirety by reference for all purposes.The method is allowed The quantitative analysis that heavy chain is matched with specific light chain is carried out in the protein mixture of coexpression, and can be used to determine when weight When chain and light chain are co-expressed, specific heavy chain immunoglobulin whether with the selection of one of two light chain immunoglobulins Property associate.The method is summarized as follows：At least one heavy chain and two different light chains are made so that heavy chain is to limit match reaction The ratio of thing is co-expressed in cell；Optionally from cell separation secretory protein；Separate from remaining secretory protein and combine heavy chain Light chain immunoglobulin polypeptide, to generate detached heavy chain pairing fraction；Determine different per bar in detached heavy chain fraction The amount of light chain；And in the heavy chain fraction of Analyze ＆ separate the light chain different per bar relative quantity, with determine at least one heavy chain with The ability of one light chain selective matching.

Rational flux is this method provided, is that stably (that is, to operation, the minor variations of such as user or flow velocity are not It is sensitive) and accurately.This method provide the sensitive analysis of the effect of the little change that can determine protein sequence.Large surface region Scrambled proteins matter-protein；Domain-domain；Chain-chain interacts generally needs multiple mutation (replacing), to introduce It is selective.Protein is not required to be separated and purifying, this allow that more effectively screening.With regard to the embodiment of the method In addition details are described in embodiment.

The replacement assay method of preferential pairing

The alternative of the preferential pairing of detection includes quantitatively including every light chain using LC-MS (liquid chromatography-mass spectrography) With respect to heterodimer colony, using the every kind of different species of the differential identification of its molecular weight.Antigenic activity analysis can also be used for Quantitatively comprising the relative heterodimer colony of every light chain, the combination degree for thus determining (relative to control) can be used to estimate Every kind of respective heterodimer colony.

Other method such as SMCA is described in embodiment, accompanying drawing and form.

Heat endurance

The heat endurance of heterodimer can be determined according to methods known in the art.The melting temperature of each heterodimer Degree is the sign of its heat endurance.The melting temperature of heterodimer can use the technology of such as differential scanning calorimetry to determine (Chen etc., (2003) Pharm Res 20：1952-60；Ghirlando etc., (1999) Immunol Lett 68：47-52). Or, the heat endurance of heterodimer can determine (Murray etc., (2002) J.Chromatogr Sci using circular dichroism 40：343-9).

The affinity of antigen

Heterodimer can be ripe according to this area with the binding affinity of respective antigen and the dissociation rate of interaction The method known is determined by competitive binding analysis.One example of competitive binding analysis is radioimmunoassay, including Be incubated in the case where the amount of unlabelled antigen increases labelled antigen (for example, 3H or 125I and molecule of interest for example this Bright heterodimer), and detect the molecule for being bound to tagged ligand.The heterodimer of the present invention and the affinity of antigen And dissociation rate can be from the saturation data determination of Scatchard analyses.

The kinetic parameter of heterodimer as herein described also can use known in the art based on surface plasmon The analysis (for example, BIAcore dynamic analyses) of resonance (SPR) is determined.Based on the commentary of the technology of SPR, referring to Mullet etc., 2000, Methods 22：77-91；Dong etc., 2002, Review in Mol.Biotech., 82：303-23；Fivash etc., 1998, Current Opinion in Biotechnology 9：97-101；Rich etc., 2000, Current Opinion in Biotechnology 11：54-61.In addition, the method for the present invention contemplates United States Patent (USP) No.6,373,577,6,289,286, 5,322,798th, described in 5,341,215,6,268,125 any SPR instruments and mutual for determining protein-protein The method based on SPR of effect.As it is known in the art, FACS can also be used for determining affinity.

The library for being mutated design collection using bispecific antibody generates bispecific antibody by Mab1 and Mab2.

In one embodiment, bispecific antibody mutation design collection is described, it is intended to from two kinds of classical antibody Mab1 With Mab2 (being made up of Fab Fab1 and Fab2 respectively) selectively formed bispecific antibody.The design collection by Fab1, Fab2 and Fc distinguish corresponding homeotic mutation and constitute.In one embodiment, design collection library is with table 5, table 12 or table Any one of 15 to the 17 design set representations for including.In the case of the light chain and heavy chain that there is competition Fab2, will be mutated The light chain of Fab1 and the interface of heavy chain are introduced, to realize that the selection between two obligate chains is matched.Pairing is selected in interface On the basis of space, hydrophobic or electrostatic complementarities between some focus Framework residues, by two obligate light chains and heavy chain Introduce favourable complemented mutant to realize, while being related to the unfavorable interfacial interaction of these Mutated residues and non-obligate chain pair. Concentrate in each design, in the case of the light chain and heavy chain that there is competition Fab1, pairing mutation also can will be selected to introduce Fab2 Light chain and heavy chain interface, with realize the selection between this two obligate chains match.The mutation is intended to reduce the light chain of Fab1 With the mispairing of the heavy chain (vice versa) of Fab2.Mutation is introduced into Fc interfaces, to realize that the selection of heavy chain is matched, so as to form bag Asymmetric antibody molecule containing two different heavy chains.Engineering at the light chain of antibody and some interface residue positions of heavy chain changes Make and generally can cause adverse effect, antigen-binding affinity, stability, solubility, aggregation tendency of the antibody etc. are lost. Many relevant natures can be affected, such as kon and koff speed, melt temperature (Tm), stress state as acid, alkali, oxidation, The stability of freeze/thaw, stirring, pressure etc..This is generally affected by the complementary determining region (CDR) of antibody of interest.Assume What the CDR of different antibodies was typically differed, then the impact of the above-mentioned property of mutation design set pair can not be in all antibody Identical.The antibody spline structure created relative to other incorrect pairings containing pollutant is there is provided herein, with notable purity Bispecific antibody, the method so as to obtain any two available antibodies Mab1 and Mab2.Introducing each mutation design collection Homeotic mutation after, make the light chain and heavy chain coexpression of Mab1 and Mab2, the antibody products expressed by Analysis and Screening are excellent to estimate The bispecific antibody of choosing, relative to the purity of other Mab sample species expressed in protein.In some embodiments In, Analysis and Screening operation can be based on LC-MS technologies.In some embodiments, Analysis and Screening operation can be based on according to electric charge point From such as capillary isoelectric focusing (cIEF) technology, or chromatographic technique.It is provided in based on the example of the triage techniques of SMCA operations In embodiment 9.In some embodiments, the notable purity of bispecific antibody is defined as in expressed protein More than the 70% of the Mab sample species of all acquisitions.In some embodiments, the notable purity of bispecific antibody is defined as In expressed protein more than all acquisitions Mab sample species 90%.Prepare and select bispecific Mab designs Collection, operation such as Figure 12 for obtaining Mab1 and Mab2 schematically shows.

Pharmaceutical composition

The present invention also provides the pharmaceutical composition comprising heterodimer as herein described or heterodimer pair.Such group Heterodimer of the compound comprising therapeutically effective amount or heterodimer pair and pharmaceutically acceptable carrier.In specific embodiment party In formula, term " pharmaceutically acceptable " means by federal regulator or state government's approval or in American Pharmacopeia Or other well known pharmacopeia are listed for animal, more particularly people (U.S.Pharmacopeia).Term " carrier " refer to Diluent, assistant agent, excipient or medium that treatment is applied together.Such pharmaceutical carrier can be sterile liquid, such as water and Oil, including those oil of oil, animal, plant or synthesis source, such as peanut oil, soybean oil, mineral oil, sesame oil etc.. When the intravenous administration of pharmaceutical composition, water is preferred carrier.Saline solution and aqueous dextrose and glycerite also may be used As liquid-carrier, solution is especially injected.Suitable drug excipient includes starch, glucose, lactose, sucrose, gelatin, wheat Bud, ground rice, chalk, silica gel, odium stearate, glycerin monostearate, talcum, sodium chloride, skimmed milk power, glycerine, propane diols, Water, ethanol etc..If desired, composition can also be comprising micro wetting or emulsifying agent or pH buffer.These compositions can be adopted Take the form of solution, suspension, emulsion, tablet, pill, capsule, pulvis, sustained release preparation etc..Composition can be formulated as tool There is the suppository of traditional base-material and carrier such as triglycerides.Oral formulations may include standard vector, the sweet dew of such as pharmaceutical grade Sugar alcohol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate etc..The example of suitable pharmaceutical carrier such as by " Remington ' s Pharmaceutical Sciences " is obtained described in E.W.Martin.Such composition will have comprising treatment The compound (preferably pure form) and the carrier of appropriate amount of effect amount, to be suitably applied in the form of patient.The system Agent should be adapted to mode of administration.

In certain embodiments, the composition comprising heterodimer or heterodimer pair is prepared according to normal process steps To be suitable to the intravenous pharmaceutical composition for being applied to the mankind.Generally, to be dissolved in sterile isotonic aqueous for the composition of intravenous administration The solution of buffer solution.When necessary, composition may also include solubilizer and local anesthetic such as lidocaine (lignocaine), alleviating the pain of injection site.In general, composition individually or is mixed and carried with unit dosage forms For the freeze-dried powder or without the form of aqueous concentrate in the unit dosage forms such as sealing container, the sealing container such as indicates activator The ampoule bottle or pouch of amount.When composition is applied by being transfused, the transfusion comprising pharmaceutical grade sterilized water or salt solution can be used Bottle makes up a prescription.When composition is applied by injecting, it is possible to provide the ampoule bottle of Injectable sterile water or salt solution, so that composition can be Mixing before applying.

In certain embodiments, composition as herein described can be formulated as neutrality or salt form.It is pharmaceutically acceptable Salt includes those pharmaceutically acceptable salts formed with anion, such as from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid Deng those anion, and with cation formed those pharmaceutically acceptable salts, such as from sodium, potassium, ammonium, calcium, Those cations of iron hydroxide isopropylamine, triethylamine, 2- ethyl amido alcohols, histidine, procaine etc..

The amount of composition as herein described will be efficiently used for the treatment of the related disease of unconventionality expression or illness, suppress and Prevention and/or the activity for the treatment of albumen, the amount can be determined by standard clinical techniques.Additionally, analyzed in vitro is optionally used Optimal dose scope is identified in help.Exact dose for preparation also relies on the serious of method of administration and disease or illness Property, and should be determined according to the situation of the judgement of practitioner and each patient.Effective dose is from from external or animal mould The dose-effect curve of type test system is inferred.

The use of heterodimer pair

As described above, heterodimer pair as herein described can include the first heterodimer and the second heterodimer, Wherein the heavy chain immunoglobulin and/or light chain immunoglobulin of each heterodimer includes known treatment antibody or combination point The one or more modifications of the known antibodies of son.It is therefore contemplated that the heterodimer of the modification comprising these antibody can be used to control Treat or prevent and known treatment antibody or known antibodies identical disease, illness or infection can be used.

In another embodiment, heterodimer as herein described to also can advantageously with it is known in the art other Therapeutic combination is used for treatment or pre- anti-cancer, autoimmune disease, inflammatory conditions or infectious diseases.In specific embodiment In, heterodimer pair as herein described can be with monoclonal or chimeric antibody, lymphokine or hemopoieticgrowth factor (such as example Such as, IL-2, IL-3 and IL-7) it is applied in combination, such as quantity for increase with the effect daughter cell of interaction of molecules or work Property, and increase immune response.Heterodimer as herein described to also can advantageously with one or more medicine, such as Anticancer, antiinflammatory or antivirotic are combined for treating disease, illness or infection.

Kit

The present invention additionally provides including the kit of one or more heterodimer pair.The single component of the kit can Pack in a separate container, and such container is accompanied by the political affairs of the manufacture, use or sale of supervision medicine or biological product The points for attention of the regulation of mansion mechanism, show the approval for obtaining manufacture, use or sale mechanism.The kit is optionally wrapped The explanation or guidance of the using method containing general introduction heterodimer pair or application program.

When one or more component of the kit is provided with the solution such as aqueous solution or aseptic aqueous solution, container means Itself can be inhalator, the injection that solution can be applied to or be applied to experimenter and mix with the other components of kit Device, suction pipe, eye dropper or other such similar devices.

The component of the kit can also be dried or lyophilized form is provided, and the kit can be additionally comprised for freezing The suitable solvent that component is restored.Quantity regardless of container or type, the kit of the present invention may also include help will combination Thing is applied to the instrument of experimenter.This quasi-instrument can be inhalator, spray nose equipment, syringe, suction pipe, operating forceps, measuring spoon, drop Means of delivery is checked and approved in eye pipe or similar medical treatment.

It is computer-implemented

In one embodiment, computer includes being connected at least one processor of chipset.It is connected to chipset Also have memory, storage device, keyboard, EGA, pointer device and network adapter.Display is connected to figure and fits Orchestration.In one embodiment, the function of chipset is provided by control centre and I/O control centres is stored.In another reality In applying scheme, memory is directly connected to processor rather than chipset.

Storage device is any equipment that can keep data, such as hard disk drive, compact disc read-only memory (CD-ROM), DVD or solid storage device.Memory keeps the instruction and data that processor is used.Pointer device can be mouse, trace ball Or other kinds of pointer device, and be used to enter data into computer system with keyboard combination.EGA is showing Display image and other information on device.Computer system is connected to LAN or wide area network by network adapter.

As it is known in the art, computer can have and different and/or miscellaneous part described above.Additionally, computer can Lack some parts.Additionally, storage device can be that computer is local and/or long-range (is such as included in storage area network In network (SAN)).

As it is known in the art, computer is able to carry out computer program module, to provide function as herein described.As herein Used, term " module " refers to the computer program logic for providing the function of specifying.Therefore, module can be with hardware, firmware And/or software is implemented.In one embodiment, program module is stored in storage device, is loaded into memory and by processing Device is performed.It should be appreciated that example as herein described and embodiment are only for schematically being illustrated, and carry out according to it Various modifications or change will make prompting to those skilled in the art, and be included in the spirit and scope of present patent application with And in scope of the following claims.

Embodiment

Hereafter it is carried out the example of specific embodiments of the present invention.Being provided only for these embodiments is carried out schematically Explanation, it is not intended to limit the scope of the present invention by any way.Make an effort to guarantee with regard to numeral used (for example, Amount, temperature etc.) accuracy, should allow the presence of some experimental errors and deviation certainly.

Except as otherwise noted, enforcement of the invention is by using protein chemistry in the art, biochemistry, recombinant DNA Technology and pharmacology conventional method.Such technology is fully explained in the literature.See, for example, T.E.Creighton, Proteins：Structures and Molecular Properties (W.H.Freeman and Company, 1993)； A.L.Lehninger, Biochemistry (Worth Publishers, Inc., latest edition)；Sambrook etc., Molecular Cloning：A Laboratory Manual (second edition, 1989)；Methods In Enzymology (S.Colowick and N.Kaplan is compiled, Academic Press, Inc.)；Remington ' s Pharmaceutical Sciences, the 18th edition (Easton, Pennsylvania：Mack Publishing Company, 1990)；Carey and Sundberg, Advanced Organic Chemistry, the 3rd edition (Plenum Press), A and B volume (1992).

Embodiment 1：Molecule modeling and computer-directed Fab interface engineerings are transformed

Structure and the method for calculating molecule modeling guiding are used to generate the library of heavy chain and light chain mutant design, the library Can screen in other antibody (Ab) or the scope of its fragment, required specificity is shown in antibody of interest to identify Mutation.The layout strategy of engineered preferential heavy chain (H)-light chain (L) pairing includes identifying representativeness Fab (i.e. first D3H44)。

As shown in table 1, the key criterion of the Fab is：Fab is people/humanized, with conventional VH and VL subgroups, and And comprising minimum framework region mutation.Additionally, structure is thought of as：Angle should be close to the average of observed antibody between VH: VL domain Value.After Fab D3H44 are selected, the computer simulation analysis at Fab interfaces are carried out using two way method, to identify and understand counterweight The important residue of the interphase interaction of chain and light chain.

First method is related to the global analysis of the variable sequence conservations and constant interface between of Fab, and the method is by Knowing the sequence and structure alignment of antibody is carried out.The comparison of the constant and variable domain sequence of various antibody sub-populations is as shown in Figure 1. Figure 1A shows the comparison of representative people VH germline subgroups.Figure 1B shows the comparison of representative people κ VL germline subgroups.Figure 1C shows the comparison of representative people λ VL germline subgroups.Fig. 1 D show the comparison of people's CH1 allelic sequences.Fig. 1 E show The comparison of people's κ and λ allelic sequences is gone out.Second method is directed to use with many molecule modeling instruments and carries out D3H44 crystal knots The analysis at structure interface, (such as ResidueContacts as shown in Figure 2^TM).The result of these analyses is identification for engineered The hotspot location list of preferential H-L pairings.The hotspot location that the analysis determines is listed in Table 2 below.These positions and amino acid master If Framework residues (in addition to a few locations in CDR3 rings), and be also substantially conservative in λ L chains.Mother body D 3H44 The amino acid of sequence and Kabat numberings are provided in table 3a-3b.

Then, hotspot location and the mutation of the possibility at hotspot location of interest pass through meter in 3D crystal structures Calculation machine simulates mutagenesis and Zymepack^TMAccumulation/modeling Simulation and identification.Zymepack^TMIt is that to provide input structure and one group prominent Become, the residue type in input structure is changed according to the mutation for being provided, and generate the physical arrangement for being similar to mutain The software kit of new construction.In addition, Zymepack assesses the property of mutain by calculating many quantitative measurements.These tolerance bags The measured value of space and electrostatic complementarities is included, the measured value is special with the stability of mutain, binding affinity or heterodimer It is different in nature related.

Fig. 3 provide variable domains in heavy chain and light chain interface hotspot location subset, and illustrate how by Mutation is introduced at these interface locations, is matched with the selection for promoting obligate chain, while repelling the formation of incorrect chain pair.Using bag Include Zymepack^TMComputational methods spatial complementary is modeled, and according to capacity factor, such as Van der Waals is piled up, hole The close contact of effect and hydrophobic grouping is calculated.Similarly, electrostatic interaction energy amount is modeled, and according to electricity Coulomb interactions, hydrogen bond and desolvation effect between lotus are estimated.Simulation is obtained by introducing mutation of interest Preferred heavy chain and light chain to model (such as H1: L1 (or H2: L2)) and it is incorrect to model (such as H1: L2 (and H2: L1 both)), to calculate space and electrostatic score.This allows whether the specific catastrophe set of determination causes favourable energy, i.e., It is right relative to incorrect (non-obligate), preferred (obligate) heavy chain-light chain pair, with bigger space and/or electrostatic complementarities.Meter Calculation space and the component that electrostatic energy is the light chain free energy related to heavy chain pairing.Therefore, bigger space and electrostatic complementarities It is the pairing relative to non-obligate pair, the bigger sign of the related Gibbs free of the pairing of obligate pair.Bigger space or quiet Electric complementary preferential (selectivity) for producing obligate heavy chain and light chain match (relative to it is non-it is obligate to).

Embodiment 2：The selection and description of design

The method of the description of embodiment 1 goes out the heavy chain-light chain heterodimer of selective or preferential pairing for design show To (such as H1-L1 and H2-L2).Heterodimer is designed in pairs, referred to as " is designed " or " design collection ", and is preferentially matched somebody with somebody including promotion To H1, L1, H2 or L2 chain on one group of displacement (table 5).Design collects with " LCCA designs " initial testing (table 4), wherein one Heavy chain is co-expressed with two light chains, to assess opposing pairs.Put with reference to table 3a, 3b identification amino acid using Kabat numbering systems Change.

The design library that the table 30 of international patent application No.PCT/CA2013/050914 is described is used as shown in identification table 4 Some LCCA design and table 5 shown in design collection starting point.Some designs in table 4 and table 5 are new independent designs.Table 6 show core design, and the unique identifier of correlation.Constant region is only crossed in major part design, and minority design also includes can Become the modification in area.The proposition of these designs is further intended to drive pairing specificity, while also helping other antibody forming systems Transferability.

For derivative design, the design library that the table 30 of international patent application No.PCT/CA2013/050914 is described is used Make starting point, the design by structural similarity cluster, and the intensity according to pairing specificity, to the effect of antigen binding with And the stability sequence determined by differential scanning calorimetry (DSC).Then combination is (referring to the example in table 7) and/or optimizes (referring to the example in table 8 and table 9) design obtains derivative design.For at least one of combination, design show high pairing Specificity, other design shows go out a series of favourable pairing specificity.All design shows for being chosen as combining and/or optimizing Go out to antigen binding without effect/least action, and to melt temperature (Tm) without effect/least action.

Individually (it is classified as independence under the kind of design row of table 5) and combines derivative design (in the kind of design of table 5 Independent/combination is classified as under row；Referring also to the example in table 10) test independent design.

Design is piled into the molecular model and computation measure (as described in Example 1) of D3H44.Then it is (right according to risk Stability and immunogenic possible impact) and affect (consider to drive and match specific suggestion intensity) to select optimum to set Meter.These optimal designs are as shown in table 5.

Embodiment 3：The preparation of the Fab constructs of encoding D 3H44IgG heavy chain and D3H44IgG light chains.

The wild type Fab heavy chain of anti-tissue factor antibodies D3H44 and light chain are prepared as described below.D3H44Fab light chains (AJ308087.1) and heavy chain (AJ308086.1) sequence takes from GenBank (table 3c), gene chemical synthesis are simultaneously expressed mammal Carry out codon optimization.Light chain vector insetion sequence is by 5 '-EcoRI cleavage site-HLA-A signal peptide-HA or FLAG labels-light Chain Ig clone-" TGA terminations "-BamH1 cleavage sites -3 ' constitute, be connected to pTT5 carriers (Durocher Y etc., Nucl.Acids Res.2002；30, No.2e9).Carrier+insetion sequence sequencing to gained, to confirm reading frame and coding The sequence of DNA is correct.Equally, heavy chain vector insetion sequence by 5 '-EcoR1 cleavage sites-HLA-A signal peptides-heavy chain clone ( T238 terminates；Referring to table 3a)-ABD₂-His₆Label-TGA terminates-BamH1 cleavage sites -3 ' and constitutes, and is connected to pTT5 carriers (ABD；Albumin binding domain).Same carrier+insetion sequence sequencing to gained, to confirm reading frame and coding DNA Sequence is correct.The Fab D3H44 constructs of the various amino acid replacements comprising design collection are given birth to by gene chemical synthesis or direct mutagenesis Into (Braman J, Papworth C＆Greener A., Methods Mol.Biol. (1996) 57：31-44).

It is preferential by competition analysis-SPR screening assessments to be conducive to respectively in C- and N- end marks heavy chain and light chain Pairing.ABD₂-His₆Heavy chain label specificity allows H-L compounds to capture in anti-his labels SPR chip surfaces, and FLAG and HA light chains label allows quantitative relative L1 and L2 colonies.

Embodiment 4：The modification of constant domain comprising D3H44IgG light chains and/or heavy chain or constant and variable domains are repaiied The preferential pairing assessment of the Fab heterodimers of decorations combination.

Encoding D 3H44IgG heavy chain comprising the amino acid modified Fab forms that collection is designed according to the LCCA of table 12 and light chain Construct prepare as described in Example 3.In the scope that LCCA designs collection (H1, L1, L2), preferentially pairing forms institute to construct The ability of the H1-L1 heterodimers for needing is determined using light chain competition analysis (LCCA).

The opposing pairs of the quantitative heavy chain of LCCA and at least light chain of two uniquenesses, and can be summarized as follows.One D3H44 heavy chain Fab constructs are co-expressed with the D3H44 light chain Fab constructs of two uniquenesses, the specific (example of opposite light pairing Such as H1-L1: H1-L2) with competition analysis-SPR screening test, repeat.(it is designed as preferentially matching somebody with somebody with H chains by reducing L1 It is right) relative to the amount (such as L1: L2=1: 3, by weight) of L2, while keep limited amount heavy chain (i.e. H1: L1+L2 is 1: 3) LCCA, is made to screen rate-distortion, to identify strong driving.The amount (i.e. H1-L1 and H1-L2) of each heterodimer for being formed Measure as described below：Heavy chain is set to be bound to SPR chips by the way that his- labels are drop-down, then using the antibody of these label specificity Detect the amount of each light chain label (HA or FLAG).Subsequently, heterodimer hit is selected by light chain competition analysis inspection, according to This L1: L2DNA ratio changes during transfecting with 1: 3 and 1: 9, while keeping limited amount heavy chain.It is also noted that in D3H44 In system, light chain label (HA or FLAG) does not affect LCCA to match (referring to international patent application No.PCT/CA2013/050914 Embodiment 10).Schematically showing for analysis design is as shown in Figure 4.Fig. 5 illustrates how to mark heavy chain and light chain and how The preferential pairing of assessment.The Experimental detail of LCCA is provided below.

Transfection method

LCCA comprising a heavy chain and two light chains construct designs are prepared as described in Example 3, transfection as described below To CHO-3E7 cells.CHO-3E7 cells are with 1.7-2x10⁶The density of individual cell/ml at 37 DEG C, be supplemented with 4mM glutamine With the FreeStyle of 0.1%Koliphor P188 (Sigma#K4894)^TMF17 culture mediums (Invitrogen cat#A- 1383501) culture in.Using PEI-pro (Polyplus transfection#115-375), with 1: 2.5 DNA: PEI ratio Rate, to the cumulative volume of 2ml 2 μ g DNA altogether are transfected.DNA-PEI mixtures are added after twenty-four hours, and cell is transferred to 32℃.Analyzed by non-reduced SDS-PAGE at the 7th day, then band is observed with Coomassie blue stain, to test supernatant Expression.H: L ratio is as shown in table 11.

Competition analysis SPR methods

In LCCA designs, the degree that D3H44 light chains are preferentially matched with D3H44 heavy chains is last using the N- positioned at every light chain The reading based on SPR of unique epitope tag at end is assessed.

Surface plasmon resonance (SPR) supplier.GLC sensor chips, Biorad ProteOn amine coupling reagent boxes (1- ethyl -3- (3- dimethylaminopropyls) carbodiimide hydrochloride (EDC), NHS (sNHS) and Monoethanolamine) and 10mM sodium acetate buffers purchased from Bio-Rad Laboratories (Canada) Ltd. (Mississauga, ON).4- (2- hydroxyethyls) -1- piperazine ethanesulfonic acids (HEPES) buffer solution, ethylenediamine tetra-acetic acid (EDTA) and NaCl are purchased from Sigma-Aldrich (Oakville, ON).The solution of 10%Tween 20 is purchased from Teknova (Hollister, CA).

The mutual thing sensing analyses of SPR.The analysis of all surface plasmon resonance uses BioRad ProteOn XPR36 instruments (Bio-Rad Laboratories (Canada) Ltd. (Mississauga, ON)) and PBST running buffer (PBS Teknova Inc, containing 0.05%Tween20) carry out at a temperature of 25 DEG C.Anti- five His captures surface uses GLM sensor cores Piece is generated, and the sensor chip is by the standard BioRad sNHS/EDC solution of 1: 5 dilution with 100 μ L/min along analyte (water It is flat) direction injection 140s activation.After activation, immediately with the flow velocity of 25 μ L/min along 25 μ g/ of analyte (vertical) direction injection ML is dissolved in anti-five His antibody (Qiagen Inc.) solution of 10mM NaOAc pH4.5, until fixed about 3000 resonance list Position (RU).By injecting 1M monoethanolamines 140s quenchings along analyte direction with 100 μ L/min, this also assures that life to remaining active set Into the simulation activation intermediate point for blank reference.

Screening with reference to anti-FLAG (Sigma Inc.) and anti-HA (Roche Inc.) monoclonal antibody heterodimer with Two steps are carried out：Indirectly capture on anti-five His surfaces, then injects anti-along analyte direction to make heterodimer along part direction FLAG and anti-HA.First, 30s of PBST is injected along part direction with 100 μ L/min, for steady baseline.For different every time Source dimer capture, 4% is diluted in PBST by unpurified heterodimer in cell culture medium.With the stream of 25 μ L/min Speed injects one to five heterodimer or control (i.e. comprising 100%HA- light chains or 100% simultaneously along single ligand channel The control of FLAG- light chains) 240s, the saturation heterodimer capture of about 300 to 400RU is obtained on anti-five His surfaces.Such as Fruit needs, and the first ligand channel is left a blank, as blank.After the heterodimer capture step, immediately along analysis object space 120s two is each injected with 50 μ L/min with steady baseline, the then anti-HA of 5nM anti-FLAG and 5nM to buffer injection twice is carried out It is secondary, wherein dissociation stage 180s, obtains the combination sensing figure of the buffer solution reference of one group of heterodimer containing each capture. Tissue factor (TF) antigen of heterodimer combination is injected in last remaining analyte channel as active control.Heterologous two Aggressiveness is regenerated by the phosphatase 11 8s of 100 μ L/min pulses 0.85%, to prepare the anti-five His surfaces of injection circulation next time.To pass Sense figure is compared and carries out dual reference using buffer blank injection and intermediate point, using ProteOn Manager softwares Sensing figure obtained by v3.0 analyses.

As a result

LCCA results are as shown in table 12,13a and 14a.It is noted that in table 13 and 14, " unique identifier " can not be with Table 5 is accurately corresponding, because the unique identifiers of two composition LCCA can be along any one direction ((collection #H1L1L2- collection # H2L2L1) or (collection #H2L2L1- collection #H1L1L2)).The last 3 row institute of the assessment of the preferential pairing of each LCCA design such as table 12 Show.Identical data are additionally included in the design of the 5th, 6 and 8 of table 13a and 14a or 10,11 and 13 row in scope.It is only to each Special H1, L1 and L2 mutation (LCCA designs) component is with unique number or ' collection # ' (such as 9567 or 9087).When data are with H1 When L1 H2 L2 forms (Fab is to form or design collection) are presented, so as to such design collection is compiled with the collection including two composition LCCA " unique identifier " of number (such as 9567-9087) is represented.It is noted that major part LCCA experiments are to comprising positioned at constant domain In the construct of interchain Fab disulfide bond (H/C233-L/C214, Kabat number) carry out.In table 13 (a and b) and 14 (a and b) In, show the success being specifically designed relative to preferential pairing, two complementations LCCA collection (H1, L1, L2 and H2, L2, L1) to highlight Represented in the form of Fab pair.Presence (the L chains of label：HA and FLAG, H chain：ABD₂-His₆) do not affect the expection of D3H44WT～ 50%: 50% neutral pairing.

In table, the LCCA data reported are that the intermediate value (H1-L1: H1-L2 and H2-L2: H2-L1) of rate form (is returned One turns to 1: 1 L1: L2DNA ratio).Additionally, LCCA data normalizations be 100% because according to observations the L1 of some variants and The total amount of L2 is markedly different from 100%.It is believed that total light chain percentage is inconsistent partially due in initial heterodimer Occurs variable non-specific binding during being trapped on SPR chips.Because LCCA is tested with 2 L1: L2DNA different ratios (L1: L2 is respectively 1: 3 and 1: 9) is carried out, and two LCCA normalization ratios are listed in table.It is noted that some LCCA experiments LCCA data are not reported, because the experimental data for being obtained does not meet inclusive criteria, and (such as the Fab being trapped on SPR chips is little In 100, or the LCCA total amounts of L1 and L2 fall outside 60 to 140 scope).

Table 12 lists the acquired all LCCA of data and designs (530).It is 1: 3 in view of two L1: L2DNA ratios With 1: 9, in 530 LCCA designs, 490 (92.5%) in these LCCA designs are with least 60% correct pairing (L1: L2DNA ratio is normalized to 1: 1).Remaining LCCA design include predominantly neutral LCCA designs (32/530 or 6.0%) LCCA designs (8/530 or 1.5%) of inconsistent results and is on a small quantity produced.Design shown in table 12 is mainly electrostatic (the specificity driving based on being interacted using hydrogen bond or charge-charge), some designs also include spatial complementary and/or chain Between covalent disulfide bonds.Some designs are also included for there is no native interchain disulfide bond (being formed by H/C233-L/C214) In the case of form the mutation of new disulfide bond.

For two heterodimers of design collection, table 13 (a and b) and 14 (a and b) are listed and are provided LCCA data 447 designs.Table 13a and 14a illustrate, the Computer Analogy Design of the description of embodiment 1 many different groups designs and The preferential pairing of H1-L1 rather than H1-L2 and H2-L2 rather than H2-L1 is realized in its modification.

Table 13 (a and b) lists pairing：The average LCCA performances of the Fab heterodimers of mispairing are (for H1-L1: H1- L2 and H2-L2: H2-L1, is normalized to the mean average of 1: 1 L1: L2 ratio) be at least 86: 14 those designs, and table 14 (a and b) list pairing：Those designs of the average LCCA performances of the Fab heterodimers of mispairing less than 86: 14.Each The performance of LCCA be normalized to 100% and 1: 1 L1: L2DNA ratio (as described in the embodiment above), and by scalar value ((ln (r1/f1) or ln (r2/f2)) (wherein r1 and r2 correspond respectively to test H1L1: H1L2 and H2L2: H2L1 under ratio Intermediate value, f1 and f2 correspond to respective experiment ratio) and pairing：The ratio description of the Fab heterodimers of mispairing.Each sets Meter also has related average LCCA performance scalar values (0.5 (ln (r1/f1)+ln (r2/f2))), and the value is also normalized to 100% And 1: 1 L1: L2DNA ratio (as described in the embodiment above).Further there is illustrated the scalar scope of each LCCA of design (LCCA1 and LCCA2 correspond respectively to H1L1: H1L2 and H2L2: H2L1 experiment).In 447 Mab designs, 354 (79.2%) at least 86: 14 LCCA performance mean values (table 13a and b) are shown.Design in table 13 (a and b) is always according to setting The similitude of meter is characterized as 13 clusters.Design in each cluster is according to average LCCA performances scalar value from up to minimum arrangement.

Additionally, the also graphic representation in the figure 7 of the LCCA data in table 13a.Fig. 7 shows box traction substation, and it illustrates each cluster Pairing：The average LCCA performance numbers of the Fab heterodimers of mispairing are at least 86: 14.The bottom of each case represents the one or four Divide position (Q1), it is the average LCCA performance numbers of intermediate value between minimum of a value and intermediate value so that represented most less than the value of the 1st quartile The data of little 25%.Horizontal bar inside case represents the second quartile, and it is the average LCCA performance numbers of intermediate value of cluster.Each case Top represent the 3rd quartile (Q3), it is the average LCCA performance numbers of intermediate value between maximum and intermediate value so that more than the 3rd The value of quartile represents the data of the 25% of maximum.Interquartile-range IQR is the difference between Q3 and Q1.Extend vertically in both direction Case must represent the data area of those values in Q1- (1.5*IQR) or Q3+ (1.5*IQR).Cover the horizontal bar table of case palpus Show the maximum and minimum value in the range of this.Data outside being present in box traction substation and case palpus are accredited as exceptional value, and appropriateness is abnormal It is worth representing (with the difference of Q1 or Q3 as 1.5*IQR to 3*IQR), limit exceptional value represents the (difference with Q1 or Q3 with plus sige It is different more than 3*IQR).

Embodiment 5：The amplification that biophysics is characterized

Amplify the heterodimer of the correct pairing (generally to 20m1) shown in unique identifier group (table 5), and it is as follows It is described to purify to test heat endurance and antigen binding.The heavy chain and light chain of each heterodimer is in 20ml CHO-3E7 cells Express in culture.CHO-3E7 cells are with 1.7-2x10⁶The density of individual cell/ml at 37 DEG C, be supplemented with 4mM glutamine With the FreeStyle of 0.1%Koliphor P188 (Sigma#K4894)^TMF17 culture mediums (Invitrogen cat#A- 1383501) culture in.Using PEI-pro (Polyplus cat#115-375), with 1: 2.5 DNA: PEI ratio, to 20ml Cumulative volume transfection 20 μ g DNA altogether.DNA-PEI mixtures are added after twenty-four hours, and cell is transferred into 32 DEG C.

The centrifuge cell after transfection 7 days, by high flux nickel affinity chromatography purifying from supernatant purification of heterologous dimer, such as Hereinafter described.Supernatant is in level pad (without calcium, magnesium and phenol red (HyClone^TMDulbecco phosphoric acid #SH30028.02) Salt buffer salt solution (DPBS)) in be diluted to 20-25% cell culture supernatants, Ran HouyuNi-NTA resins (Thermo Scientific#PT-88222) (also using equilibration buffer in advance) together stirring is incubated 12 hours.Then lead to Cross and resin is collected by centrifugation, be transferred to 96 hole sintered plates, with equilibration buffer solution three times, and useWash-out is slow Rush liquid (Sigma-Aldrich#H5413) wash-out.

After purification, (High Throughput Protein Express) is expressed by non-reduced high throughput protein Analyze, expressed using Caliper LabChip GXII (Perkin Elmer#760499) assessment heterodimers.Operation according to HT Protein Express LabChip User Guide (HT protein expression LabChip users' guidebooks) second editions LabChip GXII User Manual (LabChip GXII user's manuals) are carried out, and make following modification.By 2 μ l or 5 μ l Heterodimer sample (concentration range 5-2000ng/ μ l) and 7 μ l HT protein expression sample buffer (HT Protein Express Sample Buffer) (Perkin Elmer#760328) is added in 96 orifice plates (BioRad#HSP9601) individually Hole.Then heterodimer sample denaturation 15min at 70 DEG C is made.LabChip instruments use HT protein expression chip (HT Protein Express Chip) (Perkin Elmer#760499) and Ab-200 analysis setting operations.After use, use MilliQ water cleans chip, and is stored in 4 DEG C.

Embodiment 6：The thermal stability determination of Fab heterodimers is carried out by DSF.

To assess heat endurance, using differential scanning fluorescence (DSF) as from wild type, not modified heavy chain-light chain High throughput method to screening the heterodimer of all correct pairings.Heterodimer is prepared as described in Example 5.

The measure of heat endurance

Using the DSF heat endurances for determining all heterodimers pair as described below.Each heterodimer such as embodiment 5 The purifying, and it is diluted to 0.5mg/mL in DPBS (HyClone Cat#SH30028.02).For most of sample, Sypro Orange gel-colored working stock (Life Technologies Cat#S-6650) is by by 4 μ L Sypro Orange is gel-colored to be diluted to 2ml DPBS to prepare.DSF samples are by adding 60 μ L dilute in 14 μ L 0.5mg/mL protein The gel-colored working stocks of Sypro Orange released are preparing.However, for the protein less than 0.5mg/mL, each DSF Sample is by adding 60 μ L Sypro Orange dyestuff working stocks (to dilute i.e. in DPBS in the undiluted protein of 14 μ L To 1: 1500) preparing.Then 20 μ l aliquots are repeated using Rotor-Gene 6000qPCR instruments (QiaGen Inc) Carry out DSF analyses.1 DEG C is spaced from 30 DEG C to 94 DEG C and scans each sample, balance 10 seconds between each step, when waiting during beginning Between 30 seconds.Using the exciter filter and the launching filter of 610nM of 470nM, gain is 9.Led using the single order of denaturation curve Several maximums as Tm, by the software analysis datas of Rotor-Gene 6000.Using the following scheme for not changing and determining Tm values Modification is similarly prepared and analyzes remaining DSF sample：1) by being diluted to 2ml by 1 μ L Sypro Orange are gel-colored DPBS carrys out preparation work liquid storage, 2) analyze 30 μ l aliquots and 3) using 10 gain.

DSF results are as shown in table 12,13b and 14b.H1: L1Fab heat endurance is (with open country in the scope of LCCA designs Raw type compares the change of DSF values and DSF values) as shown in the 3rd and 4 row of table 12.Identical DSF value is also included within table 13b and 14b The 7th and 8 row designs in scope.For each the Fab heterodimer for wherein being repeated, during the Tm values reported are Value.Fab heterodimer Tm values relative to wild type Fab heterodimer (comprising HA labels wild type Fab construct, in Value Tm be 81.0 DEG C) Tm values comparison H1L1_dTm_dsf row in report.It is noted that for shortage native interchain disulfide bond The new Fab heterodimers of (between H chain C233 and L chain C214), do not assess and are not defined as lacking native interchain disulfide bond The H1L1_dTm_dsf values of corresponding wild type Fab.It is also noted that due to quality (such as low-yield, low strong of corresponding experiment Degree, the fluctuation between partially enclosed peak and the repetitive sequence of Fab heterodimers are more than 1 DEG C), do not report some Fab different The dimeric Tm values in source (the 17/230 of Fab heterodimers or 7.4%).For these Fab heterodimers some, report The corresponding estimated Tm values of Tm values of similar Fab heterodimers, the difference of the similar Fab heterodimers is only In the presence/absence of or identify the L chain labels (HA or FLAG) of connection.For estimated Tm values, corresponding wild type Tm value (81.2 DEG C) it is to obtain from all wild type Fab heterodimer constructs (the Fab constructs i.e. comprising HA labels or FLAG labels) Intermediate value.HA or FLAG labels are not significantly affected by the Tm values of wild type Fab heterodimer.In a word, compared with WT, Fab is heterologous Dimer shows similar Tm values.Including native interchain disulfide bond and the Fab heterodimers of DSF data can obtained In, 93% (195/209) Fab heterodimers show to reduce by 3 DEG C or less relative to WT.Additionally, impacted maximum Fab heterodimers show to reduce by 6.5 DEG C relative to WT.Table 12 lists the LCCA designs of Tm descendings arrangement.

Additionally, 13 amino acid replacements that the stability for identifying Fab heterodimers substantially improves are (referring to table 34).It is the similar Fab heterodimerics that there is no stable mutation in the Fab heterodimers including stable mutation and difference After the comparison of body, the stable mutation of identification.Heavy chain be stably mutated including A125R, H172R, L143F, Q179D, Q179E, Q39R, S188L and V190F.Light chain is stably mutated including Q124E, Q124R, Q160F, S176L and T180E.In a word, stable mutation increases 0.4 DEG C to 2.1 DEG C of stability.Heavy chain be stably mutated A125R, H172R, L143F, Q179D, Q179E, Q39R, S188L and V190F increase respectively 0.4 DEG C to 0.6 DEG C of stability, 0.4 DEG C to 2.1 DEG C, 0.4 DEG C, 0.5 DEG C to 0.6 DEG C, 0.5 DEG C to 0.8 DEG C, 1.1 DEG C to 1.6 DEG C, 0.4 DEG C to 1.2 DEG C and 1 DEG C.Light chain is stably mutated Q124E, Q124R, Q160F, S176L and T180E difference Increase by 0.4 DEG C to 0.5 DEG C of stability, 0.8 DEG C to 0.9 DEG C, 0.6 DEG C, 0.4 DEG C to 1.0 DEG C and 0.5 DEG C.

Embodiment 7：Fab heterodimer antigens affinity is determined.

The ability of assessment Fab heterodimer bind tissue factors, to determine amino acid replacement whether to heterodimer The ability of conjugated antigen has to be affected.Each Fab heterodimer is surveyed to the affinity of tissue factor as described by SPR It is fixed.

SPR suppliers.GLC sensor chips, Biorad ProteOn amine coupling reagent boxes (EDC, sNHS and monoethanolamine) Bio-Rad Laboratories (Canada) Ltd. (Mississauga, ON) is purchased from 10mM sodium acetate buffers.Contain The PBS running buffers of 0.05%Tween20 (PBST) are purchased from Teknoca Inc. (Hollister, CA).

Fab heterodimers are in batches.3 batches of A, B and C tests of the Fab heterodimers of purifying point.Batch A and B are stored in 4 DEG C about 1 month, then carry out SPR analyses, and the Fab heterodimers of the purifying of batch C are stored in 4 DEG C about 2 months, Then SPR analyses are carried out.The Fab heterodimers of batch C are represented in table 12 with the "+" after corresponding KD values.

The analysis of all surface plasmon resonance uses BioRad ProteOn XPR36 instrument (Bio-Rad Laboratories (Canada) Ltd. (Mississauga, ON)) and PBST running buffers carry out at a temperature of 25 DEG C. Anti- five His captures surface is generated using GLC sensor chips, standard BioRad that the sensor chip passes through 1: 5 dilution SNHS/EDC solution is with 100 μ L/min along the injection 140s activation of analyte (horizontal) direction.After activation, immediately with 25 μ L/min Flow velocity inject the anti-five His antibody (Qiagen that 25 μ g/mL are dissolved in 10mM NaOAc pH4.5 along analyte (vertical) direction Inc.) solution, until fixed about 3000 resonance units (RU).Remaining active set by with 100 μ L/min along analyte Direction injection 1M monoethanolamines 140s quenchings, this also assures that the simulation activation intermediate point generated for blank reference.

Screening is carried out with reference to the Fab heterodimers of TF antigens with two steps：Make Fab heterodimers indirect along part direction Then capture is injected the antigen of the purifying of 5 concentration and is once buffered simultaneously on anti-five His antibody surfaces along analyte direction The white dual reference of liquid air.First, 30s of buffer solution is injected along part direction with 100uL/min, with steady baseline.One to five Individual variant or control PBST is dissolved in the concentration of 3.4 μ g/ml, by they with the flow velocity of 25 μ L/min along single ligand channel simultaneously Injection 240s.For batch A and B, this obtains the average capture of about 1000RU on anti-five His surfaces, for batch C, The average capture of about 600RU is obtained on anti-five His surfaces.If desired, the first ligand channel is left a blank, as blank. After the capture step, immediately buffer injection twice is carried out along analyte direction, every time 100 μ L/min 30s, to stablize base Line, then injects 60nM, 20nM, 6.7nM, 2.2nM and 0.74nM antigen (TF) and buffer blank simultaneously with 50 μ L/min 120s, wherein dissociation stage 600s.Capture antibody surface continues 18s by the phosphoric acid of 100 μ L/min pulses 0.85%, and 18s is twice Regeneration, to prepare injection circulation next time.Sensing figure is compared and carried out using buffer blank injection and intermediate point double Reference again, using the sensing figure obtained by ProteOn Manager softwares v3.1 analyses.It is dual to be fitted to 1: 1 knot with reference to sensing figure Matched moulds type.The Rmax values of each antigen are normalized to the antibody capture level of each variant, and compare with 100%.

Antigen affinity (KD) value of Fab heterodimer samples is reported in table 12,13b and 14b.In LCCA designs In scope H1: L1Fab KD values (scope of KD, KD value and compared with wild type KD values intermediate value change) respectively such as table 12 Shown in the row of 5th, 6 and 7.The 3rd row (KD of H1-L1Fab heterodimers), the 4th row in table 13b and 14b are (compared with wild type The change of the KD of H1-L1Fab heterodimers), the 5th row (KD of H2-L2Fab heterodimers) and the 6th arrange (with wild type phase Than the change of the KD of H2-L2Fab heterodimer) in, identical KD value be also included within design to scope in.Only determine performance Go out the KD values of the Fab heterodimer samples of the Fab heterodimers capture of at least 100RU.With reference to wild type KD (0.157nM) Reflect the intermediate value of wherein wild type Fab heterodimer of the light chain comprising FLAG labels.Wild type Fab heterodimer (bag Containing FLAG or HA labels) show similar KD values so that it was observed that there are 2.6 times of differences between maximum and minimum of a value. In table 12,13b and 14b, the KD differences relative to wild type antigen binding affinity are shown, difference use-(log (KD) Design-log (KD) wild type) calculate so that on the occasion of expression compared with wild type, Fab heterodimers are affine to antigen binding The KD values of power reduce, and negative value represents that KD values increase.It is noted that some Fab heterodimers lack determines KD values.In these feelings In some of condition, Fab heterodimers are assessed, but SPR experiments show that the capture of Fab heterodimers is reduced (i.e. less than 100RU), Therefore can not possibly Accurate Determining KD values.For showing those Fab heterodimers similar to other Fab heterodimers (i.e. difference be only in the presence/absence of or identify the L chain labels (HA or FLAG) of connection), there is provided similar Fab is heterologous The corresponding estimated KD values of dimeric KD values (as shown in table 12,13b and 14b).Corresponding estimated wild type KD value (0.15nM) It is from all wild type Fab heterodimer constructs (the Fab constructs i.e. comprising HA labels or FLAG labels) acquisition Value.In a word, as a result show, the heterodimer (from the angle of design) of correct pairing shows to be tied similar to the antigen of wild type Close affinity (in about 2.3 times with reference to wild type affinity).

The super effect liquid phase color of the D3H44 heterodimers of the wild type marker of embodiment 8. and the heterodimer of preferential pairing Spectrum size exclusion chromatography (UPLC-SEC) collection of illustrative plates.

According to it is known in the art with embodiment 5 described by similar method expression and purify and have on heavy chain C- ends ABD2-His₆There is N- end tags (having FLAG in a construct, have HA in another construct) on label, light chain Wild type D3H44 heterodimer (heavy chain and a light chain).As described in Example 5, by His label affinity purifications Individually amplify and purify the heterodimer of preferential or correct pairing.

UPLC-SEC uses Waters BEH200SEC chromatographic columns (2.5mL, 4.6x150mm, stainless steel, 1.7 μm of particles) Carry out, the chromatographic column is set as 30 DEG C and in the Waters Acquity UPLC systems with PDA detectors.Fortune The row time includes 7min, the per injection of Hyclone DPBS/ improvement-calcium-magnesium running buffer (part No.SH30028.02) Cumulative volume 2.8mL, 0.4ml/min.By the UV absorbances monitoring wash-out in the range of 200-400nm, and extract the color under 280nm Spectrogram.Peak integration is carried out using the softwares of Empower 3.

Fig. 6 shows that (LCCA sets representative WT Fab heterodimers to (FLAG labels are included on L chains) and representativeness Meter 9735,9737 and 9740 H1L1Fab parts) design Fab heterodimers pair UPLC-SEC collection of illustrative plates.In general, with WT is compared, and design Fab heterodimers are to showing similar UPLC-SEC collection of illustrative plates.

Embodiment 9：Repair in the constant domain comprising bispecific antibody form or constant domain and variable domains The coexpression of decorations is concentrated, the preferential pairing assessment of heterodimer

Assessment heterodimer design, to determine whether they also allow the preferential pairing of bispecific antibody form. It is the Heterodimerization of the heavy chain for promoting unique in the example, the Fc areas of the total length heavy chain of each heterodimer is carried out not Symmetrical modification a so that heavy chain includes mutation T 350V, L351Y, F405A and Y407V, another heavy chain includes mutation T350V, T366L, K392L and T394W (EU numberings).

The preparation of construct：

Heterodimer design is tested in the scope of following bispecific antibody：A) D3H44/ trastuzumabs, b) D3H44/ Cetuximabs and c) trastuzumab/Cetuximab.It is noted that D3H44 is human antibody, trastuzumab is humanization Antibody, Cetuximab is the chimeric antibody being made up of human IgG1 and mouse Fv areas.According to design, coding is comprising amino acid modified The construct of D3H44, trastuzumab and Cetuximab IgG heavy chains and light chain be prepared as described below.D3H44, trastuzumab With the basic DNA sequence dna of the heavy chain of Cetuximab and light chain as shown in table 3C.D3H44, trastuzumab and Cetuximab are light Chain-ordering is prepared as described in Example 3, and except for the difference that some sequences lack label, and other sequences are marked comprising FLAG or HA Sign.D3H44, trastuzumab and Cetuximab sequence of heavy chain are prepared as described in Example 3, and except for the difference that total length heavy chain passes through institute The IgG1*01DNA sequences of attached coding hinge-CH2-CH3 domains are created, and are modified to the CH1 domains for promoting Fab heavy chains C- ends on Heterodimerization.It should be noted that typical C- terminal heavy chains lysine residue is removed, to prevent due to C- Terminal lysines shear and caused LC-MS inequality signals one (Lawrence W.Dick Jr. etc., Biotechnol.Bioeng.(2008)100：1132-43).

Analytical form (SMCA)

Heterodimer coexpression collection design preferences are assessed as mentioned below matches the ability to form bispecific antibody.Should Coexpression of the analysis based on four chains (H1 the and L1 chains of an antibody, H2 the and L2 chains of another antibody), and use mass spectrum (LC-MS) presence of the bispecific antibody that detection is properly formed.Fig. 8 provides four starting polypeptide chains of description and is not depositing In the case of preferential pairing between the heavy chain and light chain of heterodimer pair, the coexpression acquisition of these starting polypeptide chains The schematic diagram of possible product.Two total length heavy chain constructs and the light chain construct coexpression of two uniquenesses, obtain ten kinds of possibility Antibody type：H1-L1∶H1-L1、H1-L2∶H1-L2、H1-L1∶H1-L2、H2-L1∶H2-L1、H2-L2∶H2-L2、H2-L1∶ H2-L2, H1-L1: H2-L1, H1-L2: H2-L2, H1-L2: H2-L1 and H1-L1: H2-L2.H1-L1: H2-L2 species is correct The bispecific antibody (referring to Fig. 8) of pairing.Using LC-MS measure preferred kinds H1-L1 after pA purifying and deglycosylation: Opposing pairs specificity of the H2-L2 relative to other kinds of amount.If it is possible, it is unmarked to retain chain, obtain different from each other It is all Mab and incomplete antibody species of at least 50Da.When exclusion possibility of poor quality, by N- end tags (HA or FLAG) Add to light chain, distinguished with providing quality between enough species.

The analysis is related to the expression of bispecific antibody and screening step, referred to as SMCA.

Mass spectrometry method

After Protein A purification and non denatured deglycosylation D3H44 light chains are concentrated with D3H44 weights using mass spectrum assessment coexpression The preferential pairing degree of chain.Because D3H44/ trastuzumabs heterodimer only connects glycan comprising Fc N-, so only with one kind Enzyme N- glycosidase F (PNGase-F) processes the system.The sample of purifying is as described below to use PNGaseF deglycosylations：0.2U PNGaseF/ μ g antibody is dissolved in 50mM Tris-HCl pH7.0, the overnight incubation at 37 DEG C, final protein concentration 0.5mg/ mL.For D3H44/ Cetuximabs and trastuzumab/Cetuximab system, due to having in the Fab areas of Cetuximab Other N- connection glycan, so processing the system plus various exoglycosidases with N- glycosidases F.Generally, four kinds of enzymes are mixed Compound is used for the purposes：N- glycosidase F, beta galactosidase (Prozyme), β-NAG glycosides enzyme (New England Biolabs) and neuraminidase.N- glycosidase F remove Fc N- connection glycan, and exoglycosidase is by Fab N- connection glycan is trimmed to homogeneous core texture M3F (GlcNAc₂Man₃Fuc₁).It is as described below, made using four kinds of enzymatic mixtures The sample deglycosylation of purifying：0.2U PNGaseF/ μ g antibody, 0.002U α-neuraminidase/μ g antibody, 0.0001U Beta galactosidase/μ g antibody and 0.2U β-NAG glycosides enzyme/μ g antibody are dissolved in 50mM Tris-HCl pH7.0, The overnight incubation at 37 DEG C, final protein concentration 0.5mg/mL.However, in some cases, three kinds of ferment treatment (N- glycosidases F, beta galactosidase and neuraminidase) it is preferred, overlapped with the quality for avoiding sample component during LC-MS is analyzed. In the case of these, Fab glycan is reduced to slightly larger structure G0F (Man₃GlcNAc₂Fuc₁GlcNAc₂).Mixed using three kinds of enzymes Thing, using identical concentration and condition as described in four kinds of enzymatic mixtures the sample deglycosylation of purifying is made.After deglycosylation, will Sample storage carries out LC-MS analyses at 4 DEG C, then.

Deglycosylated protein example is using the HPLC systems of Agilent 1100 (by Ion Max Electrospray ion guns (ThermoFisher) are connected to LTQ-Orbitrap XL mass spectrograph (ThermoFisher Scientific)) it is analyzed by complete LC-MS.Sample (5 μ g) is expelled into 2.1x30mm Poros R2 reversed-phase columns (Applied Biosystems), and parsed using following gradient condition：0-3min：20% solvent B；3-6min：20-90% is molten Agent B；6-7min：90-20% solvent B；7-9min：20% solvent B.Solvent orange 2 A is 0.1% aqueous formic acid of degassing, and solvent B is de- Gas acetonitrile.Flow velocity is 3mL/min.Shunt after post, 100 μ L are introduced into EFI interface.Post is heated into 82.5 DEG C, will before post Solvent is heated to 80 DEG C, to improve protein peak shape.Using ThermoFisher Scientific ' s LTQ Positive Ion ESI calibration solutions (caffeine, MRFA and Ultramark 1621) correct LTQ-Orbitrap XL, and use 10mg/ MLCsI solution is tuned.Taper hole voltage (source fragmentation setting) is 40V, and FT resolution ratio is 7,500, and sweep limits is m/z 400-4, 000.LTQ-Orbitrap XL are tuned for the optimum detection of large-scale albumen (＞ 50kDa).

Will be comprising whole antibody using the modules of MaxEnt 1 of instrument controlling and DAS MassLynx (Waters) (m/z 2000-3800) and incomplete antibody (m/z 1400-2000).In brief, first in Xcalibur (Thermo Scientific spectrum) checks opening urporotein LC-MS data in module QualBrower, and using Waters offers File converter Databridge is converted to MassLynx compatible formats.The egg of conversion is checked in the spectrum module of MassLynx White matter is composed, and using the deconvolution of MaxEnt 1.Directly determine different antibodies species in each sample from the molecular weight collection of illustrative plates of gained Abundance.

The representative design of SMCA analyses

25 representative designs with high average LCCA performance numbers altogether are selected from cluster 1 to 12, in SMCA forms Test.Also have simultaneously similar mutation using similar driving, generation is selected according to the correspondence design collection for occupying similar spaces Table is designed.At least one representative design is selected from each cluster.Some clusters represented with a representative design (i.e. cluster 1,5,7, 8、10).Remaining cluster has more than a representative design because these clusters very big (i.e. cluster 2) or be made up of tuftlet (i.e. cluster 3, 4th, 6,9,11 and 12).Although the design consensus sequence similitude in each cluster, the difference of the tuftlet in cluster is least one set drive Dynamic mutation.For the cluster comprising tuftlet, from each tuftlet other representative design is selected.

The amino acid replacement of each cluster is listed in table 15 to 27, and indicates the corresponding representative amino of each cluster/tuftlet Acid displacement.For cluster 1, a design (9134-9521) is only selected to represent cluster, because these designs are utilized occupies similar spaces Similar electrostatic drive (referring to table 15).For all members of the cluster, H1 be designed to allow for electronegative displacement (L124E and Q179E) salt bridge is formed with the displacement (S176R and S131K or S131R) of L1 positively chargeds.H2 is designed to allow for the displacement of positively charged (L124R and Q179K or S186K) forms salt bridge with the electronegative displacements of L2 (S176D and T178D or T178E and/or T180E). The mispairing of H1L2 and H2L1 is to repel, and main cause is Coulomb repulsion.

For cluster 2, select two representative designs (9279-9518 and 9286-9402) to represent big cluster (referring to table 16). Design in the cluster is using the similar electrostatic drive for occupying similar spaces.For all members of the cluster, H1 is designed to allow for band The displacement (L124E and L143E or L143D) of negative electricity and L1 positively chargeds displacement (S176R and (Q124K and/or T178K) or The combination of (Q124K and Q160K)) form salt bridge.H2 be designed to allow for positively charged displacement (L124R and Q179K or S186K or S186R) salt bridge is formed with the electronegative displacements of L2 (S176D and T178D or T178E and/or T180E).The mistake of H1L2 and H2L1 With being to repel, main cause is Coulomb repulsion.

For cluster 3, select five representative designs (9338-9748,9815-9825,6054-9327,9066-9335 and 9121-9373) to represent five tuftlets in each (referring to table 17).All members of the cluster utilize H1 (L124E), L1 (S176R), H2 (L124R) and the similar electrostatic drive on L2 (S176D), it allows the shape in the heterodimer of preferential pairing Into salt bridge, and mispairing to be repel, main cause is Coulomb repulsion.To represent those for mainly driving using those constant regions Design, selects 6054-9327 designs to represent the tuftlet.In addition to these electrostatic interactions, a tuftlet is also including variable Area space drives (H1L45P and L1P44F), therefore selects to include the representative design of variable region driving representing the tuftlet (9338-9748).Another tuftlet also include two Fab heterodimers in variable region electrostatic drive (H1Q39E, L1Q38R, H2Q39R, L2Q38E), therefore select to include the representative design of variable region driving representing the tuftlet (9815- 9825).Additionally, a tuftlet being made up of so as to a representative design (9066-9335) a member include H1F122C and Engineered disulfide bond between L1Q124C.Remaining tuftlet represented with 9121-9373 is mainly utilized has other displacement The constant region of (S174R in H172T and L1 in H1) drives somewhat to change the interaction of H1L1 constant regions driving, while Also detect the effect of H172R in HC2.

It is every in selecting two representative designs (9168-9342 and 9118-6098) to represent two tuftlets for cluster 4 Individual (referring to table 18).All members of the cluster utilize H1 (L124E), L1 (S176R or S176K), H2 (L124R) and L2 (S176D) the similar electrostatic drive on, the driving allows to form salt bridge in the heterodimer of preferential pairing, and mispairing is to being Repel, main cause is Coulomb repulsion.The tuftlet represented with 9118-6098 is mainly used for total electrostatic drive excellent First match, and other tuftlets represented with 9168-9342 are also using the H1 (K228D) and L1 of the salt bridge for allowing to form other (S121K) displacement.

Only it is made up of (referring to table 19) 1 member with the cluster 5 that unique identifier 9116-9349 is represented.The design utilizes H1 (L124E), two electrostatic drives on L1 (S176R), H2 (L124R) and L2 (S176D), and H1 (A139W), L1 (F116A_V133G_L135V), the space on H2 (A139G_V190A) and L2 (V133G_L135W) drives.Therefore, for excellent The heterodimer for first matching, powered displacement allows to form salt bridge.For the Fab heterodimers of mispairing, due to Coulomb repulsion And other three-dimensional effect, its formation is to repel.

For cluster 6, each (referring to the table 20) in selecting two representative designs to represent two tuftlets.The cluster it is all Member is using the similar electrostatic drive in constant region (on the S186R on S131K, H2 on Q179E, L1 and L2 on H1 Q124E, Q160E and T180E), the driving allows to form salt bridge in the heterodimer of preferential pairing, and mispairing is to being to repel , main cause is Coulomb repulsion.Additionally, the tuftlet is also driven by different variable regions constituting.With unique identifier 9814- 9828 tuftlets for representing are using the electrostatic drive (Q39R on Q38R, H2 on Q39E, L1 and L2 on H1 in variable region On Q38E).Other tuftlets are driven using the variable region space being made up of the P44F in L45P and L1 in H1.Therefore, for The tuftlet, due to the three-dimensional effect for introducing, mispairing pair is also what is repelled.It is noted that the tuftlet is with from unique identifier The design of 9745-9075 represents that the difference of the design does not only have the Q38E on L2.

For cluster 7, a design (9060-9756) is only selected to represent cluster, because these designs are using similar electrostatic Drive (referring to table 21) with space.Total electrostatic drive is included on Q124R, H2 on L143E and Q179E, L1 on H1 Q124E, Q160E and T180E on Q179K and L2.Total space drives the F116A_ on A139W, the L1 included on H1 L135W on L135V and L2.Therefore, for the heterodimer of preferential pairing, the powered displacement in Fab areas allows to be formed Salt bridge.For the heterodimer of mispairing, due to Coulomb repulsion and other three-dimensional effect, its formation is to repel.

For cluster 8, a design (9820-9823) is only selected to represent cluster, because these designs are using similar electrostatic Drive (referring to table 22).In variable region, using the Q38E on the Q39R and L2 on Q38R, the H2 on Q39E, the L1 on H1. In constant region, using on Q124R, Q160K and the T178R on L143E, the L1 on H1, the Q179K on H2 and L2 Q124E, Q160E and T180E.For the heterodimer of preferential pairing, the powered displacement in Fab areas allows to form salt bridge, and For the heterodimer of mispairing, mainly due to Coulomb repulsion, its formation is to repel.

For cluster 9, each (referring to the table 23) in selecting two representative designs to represent two tuftlets.The cluster it is all Member is using the similar electrostatic drive in constant region (on the Q179K on Q124R, H2 on L143E, L1 and L2 on H1 Q124E, Q160E and T180E), the driving allows to form salt bridge in the heterodimer of preferential pairing, and mispairing is to being to repel , main cause is Coulomb repulsion.Additionally, the difference of tuftlet be drive in the presence/absence of variable region (L45P on H1 and P44F on L1).Therefore, for the tuftlet driven comprising variable region, due to the three-dimensional effect for introducing, mispairing pair is also to repel 's.The representative design of the tuftlet driven including variable region derives from unique identifier 9751-9065, the difference of the design it Only there is no the Q38E on L2 in place.The representative design for lacking the tuftlet of variable region displacement is 9611-9077.

For cluster 10, a design (9561-9095) is only selected to represent cluster, because these designs are utilized occupies similar sky Between similar electrostatic and space drive (referring to table 24).Total electrostatic drive includes L143E and the upper class of Q179E, L1 on H1 The Q179K on Q124R, Q124K or S131K, H2 like the position and Q124E and T180E on L2.Total space drives bag Include the V133W on the V133A and L2 on L124W, the L1 on H1.Therefore, for the heterodimer of preferential pairing, Fab areas In powered displacement allow to form salt bridge.For the heterodimer of mispairing, due to Coulomb repulsion and other three-dimensional effect, Its formation is to repel.

For cluster 11, in selecting three designs (9049-9759,9682-9740 and 9667-9830) to represent three tuftlets Each (referring to table 25).All members of the cluster drive the preferential pairing of heterodimer using electrostatic displacement.Therefore, it is right In the heterodimer of preferential pairing, the powered displacement in Fab areas allows to form salt bridge.It is main for the heterodimer of mispairing Will be due to Coulomb repulsion, its formation is to repel.For the tuftlet represented with unique identifier 9667-9830, displacement bag is had Include the electronegative displacement (L143E or L143D and Q179E or Q179D) on H1, the displacement of the positively charged on L1 (T178R or T178K), the electronegative displacement on the displacement (S186K or S186R or Q179K or Q179R) of the positively charged on H2 and L2 (Q124E).Also included for being formed with another tuftlet that the single member (unique identifier 9049-9759) of the tuftlet is represented The displacement of engineered disulfide bond.Utilized and two other tuftlet with remaining cluster that unique identifier 9682-9740 is represented The driving of similar H1 and L1；However, being driven using different constant regions H2 and L2.H2 utilizes L143R or L143K, and removes Outside Q124E displacements (total with two other tuftlet), L2 utilizes V133E or V133D.

For cluster 12, four designs (9696-9848,9986-9978,9692-9846 and 9587-9735) are selected to represent Each (referring to table 26) in four tuftlets.All members of the cluster drive preferentially matching somebody with somebody for heterodimer using electrostatic displacement It is right.Some members also utilization space drives.Two are driven with the tuftlet that unique identifier 9696-9848 is represented using electrostatic and space Person.Total electrostatic in the tuftlet is replaced by the S186K of the upper similar position of Q124R and T178R, H2 on L143E, the L1 on H1 Or the Q124E on S186R or Q179K or Q179R and L2 and T180E is constituted.Common space is replaced by H1 in the tuftlet S176L or V133Y or V133W on S188L and L2 is constituted；For the design using the V133Y on L2 or V133W, L143A or L124A is existed on H2, to accommodate large volume mutation.For the tuftlet represented with unique identifier 9692-9846, and with only The tuftlet that one identifier 9696-9848 is represented is compared, using similar electrostatic drive；For some members, using similar position Displacement T178E, rather than the T180E on L2.Additionally, the subset of the tuftlet also using similar position displacement T178Y or The similar spaces of the S176L on T178F, rather than L2 drive.The tuftlet represented with unique identifier 9986-9978 is merely with electrostatic Drive to drive preferential pairing.Similar total displacement is used for H1, L1 and H2；However, using different L2 displacements (S131E). Remaining tuftlet represented with unique identifier 9587-9735 utilizes H1 with the similar electrostatic drive on L1 (except for the difference that on L1 T178R be not used in all members in the tuftlet)；However, different electrostatic drives is used for H2 (L143R or L143K) and L2 (Q124E and V133E or Q124E and V133D).Two members in the tuftlet are also driven using similar space, and the space is driven It is dynamic to be made up of the S176L on S188L and L2 on H1.In a word, it is powered in Fab areas for the heterodimer of preferential pairing Displacement allows to form salt bridge.For the heterodimer of mispairing, due to Coulomb repulsion, its formation is to repel.Additionally, for same The design that sample utilization space drives, due to three-dimensional effect, its formation is also what is repelled.Cluster 13 is by member's 9122-9371 structure Into (referring to table 27).The design utilizes the engineered disulfide bond between H1F122C and L1Q124C, used as heterodimer The covalent driving of preferential pairing.Further, since the design also lacks native interchain disulfide bond, the formation of disulfide bond is by non-reduced Confirm with reduction PAGE gel.The design is not with SMCA formal testings；However, there is the situation of native interchain disulfide bond Under, drive (cluster 3, representative design 9066-9335) engineered disulfide bond of combined test with other constant region.

Transfection method

As mentioned below, the coexpression collection comprising two heavy chains and two light chains construct is transfected to CHO-3E7 cells. CHO-3E7 cells are with 1.7-2x10⁶The density of individual cell/ml at 37 DEG C, be supplemented with 4mM glutamine and 0.1% FreeStyle (TM) F17 culture medium (Invitrogen of Pluronic F-68 (Invitrogen cat#24040-032) Cat#A-1383501 culture in).Using PEI-pro (Polyplus cat#115-010), with 1: 2.5 DNA: PEI ratio, 50ug DNA altogether are transfected to the cumulative volume of 50ml.DNA-PEI mixtures are added after twenty-four hours, and cell is transferred into 32 DEG C and be incubated 7 days, then collect.By culture medium being collected by centrifugation and using 0.2 μM of Filter Vacuum of Steriflip to filter.So Filtered using albumin A MabSelect SuRe resins (GE Healthcare#17-5438-02) purifying as mentioned below afterwards Culture medium.The culture medium of filtration is applied to post (the Hyclone DPBS/ improvement, without calcium, without magnesium, # for using PBS balances before this SH-300028.02).Then heterodimer antibody type is washed using PBS, and in the centrifugal filters of Amicon ultra 15 100mM citrates pH3.6 wash-outs used in Ultracel 10K (Millipore#SCGP00525).Then exchanged using PBS Buffer solution, and by slide calliper rule evaluate sample, then carry out deglycosylation and LC-MS.

To assess wild type bispecific antibody system, (light chain of one of system preferentially combines two antibody forming systems Heavy chain) in intrinsic dual specificity system preferences, test one group of H1: H2: L1: L2DNA ratio in CHO expression.These Ratio attempts the natural differences of the expression between the heavy chain and light chain of two different antibodies of compensation and/or inherent pairing preference Property.For all bispecific antibody systems, the preferences (Fig. 9) observed between all test rate.It is appropriate for D3H44/ songs Monoclonal antibody system, observes the preferences to trastuzumab, i.e. D3H44 heavy chains and preferentially matches (referring to figure with trastuzumab light chain 9a).For D3H44/ Cetuximabs, observe the preferences to Cetuximab, i.e. D3H44 heavy chains preferentially with western appropriate former times list Anti- light chain is matched (referring to Fig. 9 b).For trastuzumab/Cetuximab system, the preferences to trastuzumab are observed, i.e. west Appropriate former times monoclonal antibody heavy chain is preferentially matched (referring to Fig. 9 c) with trastuzumab light chain.

For each tested in each bispecific antibody system in 25 representative designs, used H1: H2: L1: L2DNA ratios are to produce a large amount of bispecific antibody species, while the corresponding wild type bispecific with a small amount of incomplete antibody The ratio (referring to table 32a, b and c) of system.For D3H44/ trastuzumab systems, ratio used be 15 (H1), 15 (H2), 53 (L1), 17 (L1), wherein H1 and L1 refers to that D3H44, H2 and L2 refer to trastuzumab.For trastuzumab/Cetuximab System, ratio used is 15 (H1), 15 (H2), 17 (L1), 53 (L2), and wherein H1 and L1 refers to trastuzumab, and H2 and L2 is Refer to Cetuximab.For D3H44/ Cetuximab systems, ratio used be 15 (H1), 15 (H2), 53 (L1), 17 (L2), wherein H1 and L1 refer to that D3H44, H2 and L2 refer to Cetuximab.

Additionally, for D3H44/ Cetuximabs and trastuzumab/Cetuximab dual specificity system, in both direction Test design so that in a direction, test on the antibody 1 (Ab1) and antibody 2 (Ab2) of bispecific antibody system respectively Replace present on H1L1 and H2L2, in another " upset " direction, test on Ab2 and Ab1 deposited on H1L1 and H2L2 respectively Displacement (referring to table 28a and b)." _ 1 " appended by unique identifier represents such design：Wherein obtain stronger LCCA excellent First match result (referring to table 13a) heavy chain and correlation light chain be placed in one heavy chain it is weak competition its related light chain, and On the antibody of the light chain of non-other antibody." _ 2 " appended by unique identifier represent contrary " upset " direction：Wherein obtain compared with Strong LCCA preferentially matches the light chain of the heavy chain of result (referring to table 13a) and the correlation heavy chain that is placed in one and competes its phase strongly On the light chain of pass, rather than the antibody of the light chain of other antibody.For D3H44/ trastuzumab systems, only along " _ 1 " direction, test sets Meter is (referring to table 28c).

SMCA results

D3H44/ trastuzumabs system is only processed with a kind of enzyme (PNGase-F) and complete deglycosylation is carried out.For multienzyme Process, the connection sugar in Fab areas is generally punctured into core M3F (using four kinds of ferment treatments) or G0F (using 3 kinds of ferment treatments).Always It, in most of the cases, deglycosylation is processed and produces all possible different types of energy that can identify that LC-MS is identified Power.In many cases, every kind of species is represented with single LC-MS peaks.Exception includes likely corresponding to required bispecific kind The side peak (possibly adduct or the inhomogeneity of leader peptide cutting) of class；However, due to the ambiguity at side peak, in bispecific These side peaks are not considered in the contribution of species.Additionally, D3H44/ Cetuximabs (3519_1,3522_1) and trastuzumab/west Some designs in appropriate former times monoclonal antibody (9748-9338_1) system need multiple peaks to explain the ripple of the high mannose due to being connected The species moved and produce.All these designs introduce glycosylation site in Cetuximab light chain.It is noted that in certain situation Under, due to the mass separation between species relatively low (i.e. difference is less than 50Da), it is impossible to distinguish some little species (including less than institute Have species 5%).Additionally, required bispecific species H1-L1_H2-L2 generally can not be based on LC/MS by experiment and mistake Distinguish with type H1-L2_H2-L1.Therefore, when bispecific content is reported in the table, it is impossible to exclude it completely and do not include The mispairing species of the type.However, it was observed that content very low species such as H1-L2_H1-L2 and H2-L1_H2-L1 and H1-L2 and H2-L1 incomplete antibodies are the signs for micro (if any) bispecific species impurity only occur.

LC-MS data are provided in table 29a, 29b and 29c.As a comparison, wild type data are also provided in table 33a, 33b In 33c, and represented with " NA " in " SMCA unique identifiers " row and in " cluster " row.All three bispecifics are wild Type system shows the preferences of distortion so that a light chain is better than with reference to two heavy chains (referring to table 33 and Fig. 9).Additionally, extremely Less in trastuzumab/Cetuximab system, label position seems also H1L1 and H2L2 to be matched to have to significantly affect.Cause This, is to assess transferability of bispecific species of the design to needed for wild type and the impact of percentage, with identical H1: H2: L1: L2DNA ratio carries out the comparison with corresponding wild type bispecific construct, and is reported in the " change of %H1L1 pairings Change (all H1 species), relative to wild type ", " change (all H2 species) of %H2L2 pairings, relative to wild type " and " % H1: H2: L1: L2 change, relative to wild type " (only considering whole antibody species) row are (referring to table 29).It is noted that for % H1L1 matches the assessment of (all H1 species) or %H2L2 pairings (all H2 species), assesses all kinds matching somebody with somebody in Fab areas It is right.When corresponding wild type bispecific construct is not assessed by SMCA, it is compared with similar wild type construct. Estimate with report value after " * * * " represent.The similar wild type construct selected for comparing as described below.Can for assessment Metastatic, each wild type construct is tested with the SMCA for showing highest " %H1L1 and %H2L2 pairings (all kinds) " (carrying out in different ratios) represents.It is the impact for assessing bispecific species of the design to needed for and the percentage of wild type, Each wild type construct is showing highest %H1: the SMCA of H2: L1: L2 (only considering whole antibody species) is tested (with difference Ratio carry out) represent.For two kinds of situations, in all wild type constructs in dual specificity system, intermediate value is selected As the wild offset for comparing.

For each design, transferability passes through to be matched relative to overall H: L of wild type in record all kinds, especially It is the increase of all H1 species of %H1L1/ and/or all H2 species of %H2L2/ assessing.Additionally, also assessing to required double spies The impact of the percentage of different in nature species, it focuses on that only whole antibody species compares, because incomplete antibody (if present) can be by system Standby property SEC is removed/minimized by H1: H2: L1: L2DNA other titration.Table 30a, b and c show that preparative SEC can have Effect ground removing/minimizing for incomplete antibody species.Table 32a, b and c show that the percentage of incomplete antibody species also can be in the transfection phase Between using various DNA titrate ratio operation.

For D3H44/ Cetuximab systems (table 29a), shift all in addition to a design (9327-6054_2) Design, is assessed as matched by the H1L1 in all kinds relative to wild type.Major part design (except 9327-6054_2 and Outside 9134-9521_2) also show, when whole antibody species is only considered, the percentage of required bispecific antibody increases.This Outward, in addition to a design do not shifted (9327-6054_2), design reduces the initial mispairing antibody kind observed by wild type Class (H1H2L2L2).Additionally, in addition to the 9327-6054_2 and corresponding design 9327-6054_1 in other directions, designing with two Individual direction transfer, major part design shows similar effective H: L pairing in both direction.

For D3H44/ trastuzumab systems (table 29b), all designs are shifted, such as passed through in all kinds relative to wild The H1L1 pairings of type are assessed.Additionally, the percentages of bispecific antibody needed for all designs show increase (when only considering During whole antibody species).Additionally, most of design significantly reduces the initial mispairing antibody type observed by wild type (H1H2L2L2).It is noted, however, that due to lacking expression, not reporting the data (table 28c) of 9611-9077_1.

For trastuzumab/Cetuximab system (table 29c), at least 35 in 49 designs show transferable Property, such as by all H2 species in (" %H2L2 pairing change (all H2 species), relative to wild type " row on the occasion of) H2L2 pairings are assessed.The design for seeming not shifting includes 9279-9518_2,3522_2,9815-9825_2,9327-6054_ 2、9118-6098_2、9748-9338_2、9692-9846_2、9587-9735_2、9814-9828_2、3519_2、9986- 9978_2,9168-9342_2 and 9066-9335_1 (" change (all H2 species) of %H2L2 pairings, relative to wild type " row Negative value)；However, the design show in other directions goes out transferability (it is noted that due to lacking sample, not testing 9279- 9518_1).All design shows for showing transferability go out the antibody type of the observed initial mispairing of wild type experiment (H1H2L1L1) percentage is reduced.Additionally, in the design of transfer, when whole antibody species is only considered, compared with wild type, Only 2 designs (9134-9521_1 and 9279-9518_2) show that the percentage of required bispecific antibody is reduced.

In general, the design that H: L pairing of most of weak competition antibody increases makes the hundred of required bispecific antibody Divide than reducing (only considering whole antibody).For direction, compared with " _ 2 " direction, most of design show in " _ 1 " direction goes out class Like or the more preferable transferability (exception is mainly observed in trastuzumab/Cetuximab system) of H: L paired comparisons.This Outward, table 35a lists all 3 tests dual specificity system (D3H44/ Cetuximabs, D3H44/ trastuzumabs and bent appropriate lists Anti-/Cetuximab) in both direction transfer those designs.Table 35b lists all 3 dual specificity systems In a direction transfer in (D3H44/ Cetuximabs, D3H44/ trastuzumabs and trastuzumab/Cetuximab), and only In those designs of another direction transfer in one dual specificity system.Additionally, in the direction indicated, identical mutation is present Heavy chain and the same endogenous light chain of weak competition in all 3 dual specificity systems, and light chain utilization rate is at least above 10%.

For the transferability and performance of cluster, the institute for D3H44/ trastuzumab dual specificity systems, in all clusters There is member to show transferability (referring to Figure 11 a), and required bispecific antibody percentage increase (only considers complete anti- Body) (referring to Figure 11 b).For D3H44/ Cetuximab dual specificity systems, all clusters show transferability, wherein with Wild type is compared, and in all H1 species, only one member shows that H1L1 pairings are reduced (referring to Figure 11 c) in cluster 3.In addition, The member that all clusters include the bispecific antibody needed for showing and increase relative to the percentage of wild type (only considers complete anti- Body)；However, 3 clusters (cluster 1,3 and 4) also include that the bispecific antibody needed for showing subtracts relative to the percentage of wild type Few member's (only considering whole antibody) (referring to Figure 11 d).For trastuzumab/Cetuximab dual specificity system, all clusters Including the variant for showing to design transferability；However, only several clusters (1,5,7,8,10,11) including wherein all it is respective into Member shows the variant (referring to Figure 11 e) of transferability.Additionally, all clusters include that the bispecific antibody needed for showing is relative In member's (only considering whole antibody) (referring to Figure 11 f) that the percentage of wild type increases.For wherein all members show can Metastatic those clusters, all members in cluster 5,7,8,10 and 11 also show required bispecific antibody relative to wild The percentage of type increases (only considering whole antibody).

In a word, all members performance when all 3 dual specificity systems are integrally considered, in cluster 1,5,7,8,10 and 11 Go out transferability (referring to Figure 11 g)；Cluster 5,7,8,10 and 11 includes such member, wherein the bispecific needed for showing resists Body phase is for all members (only considering whole antibody) (referring to Figure 11 h) that the percentage of wild type increases.

In a word, the design that H: L pairing of most of weak competition antibody increases makes the percentage of required bispecific antibody Reduce (only considering whole antibody).For direction, compared with " _ 2 " direction, most of design show in " _ 1 " direction go out to be similar to or The transferability (exception is mainly observed in trastuzumab/Cetuximab system) of more preferable H: L paired comparisons.

Embodiment 10：The selection SMCA bispecific heterodimer antibody characterized for biophysics and parental generation Mab Preparative size exclusion chromatography (SEC).

Selecting the subset of SMCA samples carries out other biophysics sign.Most of these SMCA samples are generally showed Go out incomplete antibody species high pairing (more than～80% pairing, in H1L1+H2L2/ all kinds row) and low content (be less than- 30%, it is considered to all incomplete antibody species).Preparative SEC is proceeded as described below.Using installed in Pharmacia (GE Healthcare)The GL of Superdex 200 10/300 (GE Healthcare) post separation in Purifier systems Heterodimer antibody samples.PBS will be dissolved in (Hyclone DPBS/ are improved, without calcium, without magnesium, Cat no SH-300028.02) Heterodimer antibody samples (0.3-0.5ml) manual loading to 0.5ml rings (be filled with PBS).Then sample is noted automatically Pillar is mapped to, and post is eluted with 0.5ml/min parsings using 1CV.Monitoring OD₂₈₀Under Protein elution, and collect 0.5ml levels Point.For each SMCA sample, converge those fractions comprising main peak, and further carry out biophysics sign.

Embodiment 11：After preparative size exclusion chromatography, the bispecific heterodimer of antibody formation is preferentially matched somebody with somebody To assessment

After preparative SEC, as described in Example 9, the bispecific heterologous two for selecting sample is analyzed using LC-MS methods The preferential pairing of dimer antibody.All these samples show that the percentage of required bispecific antibody species increases, Yi Jiban The percentage of antibody type reduces (table 29 and 30).

Embodiment 12：The heat endurance of SMCA bispecific heterodimer antibody.

After preparative SEC, the heat endurance for selecting SMCA bispecific heterodimer antibody, and and parental generation are determined D3H44 and trastuzumab monoclonal antibody and Cetuximab single armed antibody are compared.In general, single armed antibody is referred to Lacked Fab areas (including C233S displacements) by a total length heavy chain, one truncates the construct that heavy chain and a light chain are constituted, Wherein heavy chain Heterodimerization is realized as described in Example 9.

The measure of heat endurance

Bispecific heterodimer antibody and the heat endurance of wild type control is selected to use differential scanning calorimetry (DSC) determine as mentioned below.After the process of preparative SEC, using VP-Capillary DSC (GE Healthcare) to master The 400 μ L samples to be dissolved in PBS with the concentration of 0.2mg/ml or 0.4mg/mL carry out dsc analysis.Start in each DSC operations When, carry out 5 buffer blanks and inject with steady baseline, and buffer injection is carried out, followed by sample injection reference every time. Filtered with 60 DEG C/h of speed and low feedback, 8s, 5min preTstat and 70psi nitrogen pressure is swept from 20 DEG C to 100 DEG C Retouch each sample.With reference to the heat score-curve of gained, and using the software analysis of Origin 7.

As a result in being shown in table 31a, b and c.The wild type Fab Tm values reported in form are from D3H44 (79 DEG C) and bent appropriate list The homodimer antibody of anti-(81 DEG C) and the single armed antibody of Cetuximab (72 DEG C) are obtained.With regard to WT D3H44/ western appropriate former times For monoclonal antibody and trastuzumab/Cetuximab heterodimer antibody, corresponding 2 peaks of Fab Tm are only observed.Do not observe To CH2 (due to overlapping from Cetuximab Fab) or CH3's (due to Chong Die with the Tm values of D3H44 and trastuzumab Fab) is different Peak.For WT D3H44/ trastuzumab heterodimer antibody, due to D3H44 and the Tm value phases of two Fab of trastuzumab Seemingly, the peak value at 81 DEG C likely corresponds to two Fab, and the peak value at about 72 DEG C likely corresponds to CH2.

In table 31a, b and c, two Fab Tm values at corresponding peak are only reported, except as otherwise noted.It is also noted that with regard to one For a little heterodimer samples, protein concentration very low (being less than 0.4mg/mL), causing the noise of baseline increases.Therefore, exist In D3H44/ trastuzumab systems, some samples produce the DSC curve of low peak intensity, making it difficult to distinguish CH2 peaks and possibility Unstable Fab.In these cases, the Tm values (table 31a) at 70 to 72 DEG C are also reported.In a word, most of heterodimer Antibody exhibits go out the heat endurance similar to corresponding wild type molecule (3 DEG C less).Additionally, most of heterodimer resists Body does not show other peak, and hint CH2 or CH3 peaks are substantially unstable.One exception is included from trastuzumab/Cetuximab system Unite engineered heterodimer antibody 9611-9077_2, and it shows other peak at 60 DEG C, it may be possible to due to CH2 not It is stable.

Embodiment 13：The antigen affinity of bispecific heterodimer antibody is determined

Assessment bispecific antibody combines the ability of related antigen, to determine whether amino acid replacement is combined with to antigen Affect.Antigen-binding affinity is determined as mentioned below by SPR.

SPR bio-sensings are analyzed

EDC：1- ethyl -3- (3- dimethylaminopropyls) carbodiimide hydrochloride；NHS：N-hydroxy-succinamide； SPR：Surface plasmon resonance；EDTA：Ethylenediamine tetra-acetic acid；TF：Tissue factor；EGFR ECD：EGF-R ELISA Extracellular domain；Her2ECD：The extracellular domain of people's epithelial growth factor receptor 2.

SPR suppliers.Series sensor chip CM5, Biacore amine coupling reagent box (NHS, EDC and 1M ethanol Amine) and 10mM sodium acetate buffers be purchased from GE Healthcare Life Science (Mississauga, ON).Restructuring Her2 Extracellular domain (ECD) albumen is purchased from eBioscience (San Diego, CA).PBS runtime buffers containing 1%Tween20 (PBST) Liquid is purchased from Teknova Inc. (Hollister, CA).Goat polyclonal anti-human Fc antibodies are purchased from Jackson Immuno Research Laboratories Inc. (West Grove, PA).EDTA is purchased from Bioshop (Burlington, ON).

The analysis of all surface plasmon resonance uses Biacore T200 surfaces plasmon resonance instrument (GE Healthcare Life Science, (Mississauga, ON)) and PBST running buffers (add 0.5M EDTA liquid storages reach To the final concentration of 3.4mM) carry out at a temperature of 25 DEG C.Anti-human Fc captures surface utilizes Series sensor chip CM5, makes With default parameters, (it is set as targeting 2000 altogether by the Immobilization Wizard of Biacore T200 control softwares Unit of shaking (RU)) generate.Screening is carried out with reference to the antibody variants of Her2ECD, TF or EGFR ECD antigenic targets with two steps：Antibody Variant captures anti-human Fc antibodies' flowing pool surface indirectly, the purifying antigen of 5 concentration is then injected, using single cycle dynamics Method carries out dynamic analysis.For capture variant or control with 1 μ g/mL on single flow cell with the flow velocity of 10 μ L/min Injection 60s.In general, as a result it is to capture about 50 to 100RU on anti-human Fc surfaces.First flow cell is left a blank, as blank Control.After the capture step, the antigen for injecting five concentration in order with 100 μ L/min on all four flow cell immediately (TF or EGFR ECD antigens be 5nM, 2.5nM, 1.25nM, 0.63nM and 0.31nM, or Her2ECD antigens be 40nm, 20nm, 10nm, 5nm and 2.5nm) 180s, the dissociation stage 300s of EGFR ECD, the dissociation stage 1800s of Her2ECD, the dissociation rank of TF Section 3600s.Capture antibody surface is regenerated by 10mM glycine pH1.5,30 μ L/min, 120s, is followed with preparing injection next time Ring.For each analyte injection, simulated cushioned liquid injection at least twice is carried out, for use as reference.Using Biacore Single cycle dynamics sensing figure obtained by T200BiaEvaluation software analysis, and it is fitted to 1: 1 binding model.

With reference to respective wild type control：Mab, the trastuzumab OAA of D3H44 and Cetuximab OAA assessments heterologous two The antigen affinity of dimer antibody.Have also obtained the antigen affinity of wild type bispecific antibody；However, WT bispecifics SPR captures can be inhomogenous (being for example related to capture the heterodimer of mispairing), so as to disturb KD to determine (referring to table 31a And c).For the heterodimer antibody for determining antigen binding in D3H44/ Cetuximab systems, antigen affinity class It is similar to corresponding WT to compare (referring to table 31b).Just determine in D3H44/ trastuzumabs and trastuzumab/Cetuximab system For most of heterodimer antibody of antigen binding, antigen affinity compare similar to corresponding WT (referring to table 31a and c).Exception includes not showing 11 engineering reform antibodies of Her2 combinations.In D3H44/ trastuzumabs and trastuzumab/west In appropriate former times monoclonal antibody system, do not observe six engineered heterodimer antibody 9049-9759_1 and 9682-9740_1 and The her2 of 3522_1 is combined.Additionally, for trastuzumab/Cetuximab system, five other engineering reform antibodies 9696-9848_1,9561-9095_2,9611-9077_2,9286-9402_2 and 9060-9756_2 also lack Her2 combinations.This The constant region on ten total H chains (L143E_K145T) and L chains (Q124R_T178R) in 11 engineering reform antibodies is dashed forward Become.The constant region mutation of identical and L chains on total H chains (L143E_K145T) of another engineering reform antibody 9286-9402_2 Similar mutation on (Q124K and S176R).

Embodiment 14：Engineered heterodimer antibody and wild type heterologous dimer and homodimer antibody Super effect liquid phase chromatogram size exclusion chromatography (UPLC-SEC) collection of illustrative plates

In the preparation of engineered heterodimer antibody and control wild type bispecific and homodimer antibody After property SEC, UPLC-SEC is entered using Waters BEH200SEC posts (2.5mL, 4.6x150mm, stainless steel, 1.7 μm of particles) OK, the chromatographic column is set as 30 DEG C and in the Waters Acquity UPLC systems with PDA detectors.Operation Time includes 7min, per injection cumulative volume 2.8mL, and running buffer is PBS and 0.02% polysorbate20 or 20mM NaPO4,50mM KCl, 0.02% polysorbate20,10% acetonitrile, pH7,0.4ml/min.In the range of 210-400nm UV absorbances monitoring wash-out, and extract the chromatogram under 280nm.Peak integration is carried out using the softwares of Empower 3.

Figure 10 shows representative engineered heterodimer antibody and representative wild type heterologous homodimeric antibody UPLC-SEC collection of illustrative plates.In most of the cases, engineered heterodimer antibody shows and corresponding wild type heterologous The similar UPLC-SEC collection of illustrative plates of homodimeric antibody, with regard to D3H44/ trastuzumabs, D3H44/ Cetuximabs and trastuzumab/western appropriate For former times monoclonal antibody, the average percent of monomer be respectively 99.18%, 98.70% and 98.77% (referring to table 31a, 31b and 31c)。

It is related although being particularly shown and described the present invention with reference to preferred embodiment and various alternate embodiments Field it will be appreciated by the skilled person that without departing from the spirit and scope of the present invention, can to form therein and in detail Feelings are variously modified.

All bibliography disclosed herein, issued patents, patent are disclosed with sequence accession number accordingly in full with the side of reference Formula is incorporated to for all purposes.

Form

Table 1：The key criterion of Fab models

Table 2：The focus amino acid position (typical Fab includes κ light chains) of the interface of heavy chain and light chain in D3H44.

Heavy chain *	Light chain *
		V37	Y36
Q39	Q38
		L45	P44
W47	L89
		F100	F98
W103	F116
		L124	F118
A139	V133
		F174	L135

* Kabat is numbered

Table 4. has the LCCA designs of the modification of a heavy chain immunoglobulin and/or two light chain immunoglobulins, its Middle H1 is preferentially matched with L1

* Kabat is numbered；WT refers to the wild-type immunoglobulin chain without amino acid mutation.

* is to each unique H1, L1 and L2 catastrophe set (LCCA forms) allocation set numbering or " unique identifier ".

Table 11：For light chain competition analysis and H1: L1: L2DNA ratio of inspection

^ other DNA：AKTdd pTT22 refer to that (dominant positivity AKT is dashed forward comprising the mutation of constitutive activity protein kinase B Become) carrier；SsDNA refers to salmon sperm DNA.

LCCA performances, stability and antigen binding assessment that the LCCA of table 12. is designed, with H1L1Fab heterodimers DSF values descending is arranged

* represent from difference to be only different in the presence/absence of other Fab of the L chain labels (HA or FLAG) of connection The dimeric estimate in source.

* values are from 333 (H1), 250 (L1), 749 (L2) LCCA experiments.

* * values are from 333 (H1), 100 (L1), 899 (L2) LCCA experiments.

ND represents that data are unavailable.

Table 13a. meets correct pairing：Mispairing Fab heterodimers are the design of 86: 14 LCCA average performance standards LCCA performances

* it is worth from LCCA experiments (being carried out with 1: 3 L1: L2DNA ratio) and obtains, and is normalized to 1: 1 L1: L2DNA ratio Rate

* values are obtained from LCCA experiments (being carried out with 1: 9 L1: L2DNA ratio), and are normalized to 1: 1 L1: L2DNA ratio Rate

* * " unique identifier " are by along (collection #H1L1L2- collection #H2L2L1) or (collection #H2L2L1- collection #H1L1L2) direction The unique identifier of two composition LCCA is constituted

Table 13b. meets correct pairing：Mispairing Fab heterodimers are the design of 86: 14 LCCA average performance standards Stability and antigen binding are assessed

* " unique identifier " is by along (collection #H1L1L2- collection #H2L2L1) or (collection #H2L2L1- collection #H1L1L2) direction The unique identifier of two composition LCCA is constituted

Table 14a. performances are less than correct pairing：Mispairing Fab heterodimers set for 86: 14 LCCA average performance standards The LCCA performances of meter

Claims (according to the 19th article of modification of treaty)

1. a kind of detached antigen-binding polypeptides construct comprising at least the first heterodimer and the second heterodimer,

First heterodimer includes the first heavy chain immunoglobulin peptide sequence (H1) and the first light chain immunoglobulin Peptide sequence (L1)；And second heterodimer is exempted from comprising the second heavy chain immunoglobulin peptide sequence (H2) and second Epidemic disease immunoglobulin light chains peptide sequence (L2), wherein at least one of described H1 or L1 sequences of first heterodimer are no It is same as the described corresponding H2 or L2 sequences of second heterodimer, and wherein

The each self-contained at least heavy-chain variable domains (V of H1 and H2_HDomain) and heavy chain constant domain (C_H1Domain)；

The each self-contained at least light variable domains (V of L1 and L2_LDomain) and light chain constant domain (C_LDomain)；And

At least one of H1, H2, L1 and L2 are amino acid modified comprising at least one, wherein H1 preferentially matches somebody with somebody with L1 compared with L2 It is right, and H2 is preferentially matched with L2 compared with L1；Or

H1, H2, L1 and L2 are amino acid modified comprising one group, wherein H1 is preferentially matched with L1 compared with L2, and compared with L1 H2 is preferentially matched with L2；

The melt temperature that the heat endurance in wherein described Fab areas passes through at least one of first and second heterodimer (Tm) determine, the melt temperature is described corresponding heterologous without described at least one amino acid modified or amino acid modified group Within about 0,1,2,3,4,5,6,7,8,9 or 10 DEG C of the dimeric Tm.

2. construct according to claim 1, wherein

I () H1 and/or H2 is amino acid modified comprising at least one or a group at L124, K145, D146, Q179 and S186, and And

(ii) L1 and/or L2 includes at least one or one group of ammonia at Q124, S131, V133, Q160, S176, T178 and T180 Base acid modification.

3. construct according to claim 2, wherein

(i) H1 and/or H2 comprising selected from L124R, L124E, K145M, K145T, D146N, Q179E, Q179K, S186R and At least one or one group of S186K is amino acid modified, and

(ii) L1 and/or L2 comprising selected from Q124E, S131R, S131K, V133G, Q160E, S176R, S176D, T178D, At least one or one group of T178E and T180E is amino acid modified.

4. construct according to claim 3, wherein：

H1 is comprising selected from the amino acid modified of L124E, K145M, K145T and Q179E or combinations thereof；

L1 is comprising selected from the amino acid modified of S131R, S131K, V133G and S176R or combinations thereof；

H2 is comprising selected from the amino acid modified of L124R, D146N, Q179K, S186R and S186K or combinations thereof；And

L2 includes the amino acid selected from Q124E, V133G, Q160E, S176D, T178D, T178E and T180E or combinations thereof Modification.

5. construct according to claim 4, wherein：

H1 includes amino acid modified L124E, K145T and Q179E；

L1 includes amino acid modified S131K, V133G and S176R；

H2 includes amino acid modified L124R and S186R；And

L2 includes amino acid modified V133G, S176D and T178D.

6. construct according to claim 1, wherein

I () H1 and/or H2 is repaiied comprising at least one or one group of amino acid at L124, L143, K145, D146, Q179 and S186 Decorations；And

(ii) L1 and/or L2 is repaiied comprising at least one or one group of amino acid at Q124, V133, Q160, S176, T178 and T180 Decorations.

7. construct according to claim 6, wherein

I () H1 and/or H2 is comprising selected from L124E, L124R, L143E, L143D, K145T, K145M, D146N, Q179K, S186R It is amino acid modified with least one or one of S186K group；And

(ii) L1 and/or L2 comprising selected from Q124K, Q124E, V133G, Q160K, S176R, S176D, T178E, T178K, At least one or one group of T178R, T178D and T180E is amino acid modified.

8. construct according to claim 7, wherein：

H1 is comprising selected from the amino acid modified of L124E, L143E, L143D, K145T and K145M or combinations thereof；

L1 is comprising selected from the amino acid modified of Q124K, V133G, Q160K, S176R, T178K and T178R or combinations thereof；

L2 is comprising selected from the amino acid modified of Q124E, V133G, S176D, T178E, T178D and T180E or combinations thereof.

9. construct according to claim 8, wherein：

H1 includes amino acid modified L124E, L143E and K145T；

L1 includes amino acid modified Q124K, V133G and S176R；

H2 includes amino acid modified L124R and Q179K；And

L2 includes amino acid modified V133G, S176D and T178E.

10. construct according to claim 8, wherein：

H1 includes amino acid modified L124E, L143E and K145T；

L1 includes amino acid modified Q124K, V133G and S176R；

H2 includes amino acid modified L124R and S186R；And

L2 includes amino acid modified V133G, S176D and T178D.

11. constructs according to claim 1, wherein

I () H1 and/or H2 is amino acid modified comprising at least one or a group at Q39, L45, L124, L143, F122 and H172, And

(ii) L1 and/or L2 includes at least one at Q38, P44, Q124, S131, V133, N137, S174, S176 and T178 Or one group amino acid modified.

12. constructs according to claim 11, wherein

I () H1 and/or H2 is comprising selected from Q39E, Q39R, L45P, F122C, L124E, L124R, L143F, H172T and H172R At least one or one group amino acid modified；And

(ii) L1 and/or L2 comprising selected from Q38R, Q38E, P44F, Q124C, S131T, S131E, V133G, N137K, S174R, At least one or one group of S176R, S176K, S176D, T178Y and T178D is amino acid modified.

13. constructs according to claim 12, wherein

H1 is repaiied comprising the amino acid selected from Q39E, L45P, F122C, L124E, L143F, H172T and H172R or combinations thereof Decorations；

L1 comprising selected from Q38R, P44F, Q124C, S131T, V133G, N137K, S174R, S176R, S176K and T178Y or it Combination it is amino acid modified；

H2 is comprising selected from the amino acid modified of Q39R, L124R and H172R or combinations thereof；And

L2 is comprising selected from the amino acid modified of Q38E, S131E, V133G, S176D and T178D or combinations thereof.

14. constructs according to claim 13, wherein：

H1 includes amino acid modified Q39E and L124E；

L1 includes amino acid modified Q38R, V133G and S176R；

H2 includes amino acid modified Q39R and L124R；And

L2 includes amino acid modified Q38E, V133G and S176D.

15. constructs according to claim 13, wherein：

H1 includes amino acid modified L45P and L124E；

L1 includes amino acid modified P44F, V133G and S176R；

H2 includes amino acid modified L124R；And

L2 includes amino acid modified V133G, S176D and T178D.

16. constructs according to claim 13, wherein：

H1 includes amino acid modified L124E and L143F；

L1 includes amino acid modified V133G and S176R；

H2 includes amino acid modified L124R；And

L2 includes amino acid modified V133G, S176D and T178D.

17. constructs according to claim 13, wherein：

H1 includes amino acid modified F122C and L124E；

L1 includes amino acid modified Q124C, V133G and S176R；

H2 includes amino acid modified L124R；And

L2 includes amino acid modified V133G and S176D.

18. constructs according to claim 13, wherein：

H1 includes amino acid modified L124E and H172T；

L1 includes amino acid modified V133G, N137K, S174R and S176R；

H2 includes amino acid modified L124R and H172R；And

L2 includes amino acid modified V133G, S176D and T178D.

19. constructs according to claim 1, wherein

I () H1 and/or H2 is amino acid modified comprising at least one or a group at L124, A125, H172 and K228, and

(ii) L1 and/or L2 is repaiied comprising at least one or one group of amino acid at S121, V133, N137, S174, S176 and T178 Decorations.

20. constructs according to claim 19, wherein

(i) H1 and/or H2 comprising selected from L124E, L124R, A125S, A125R, H172R, H172T and K228D at least one or One group amino acid modified；And

(ii) L1 and/or L2 is included selected from S121K, V133G, N137K, S174R, S176K, S176R, S176D and T178D extremely Few one or one group amino acid modified.

21. constructs according to claim 20, wherein

H1 is comprising selected from the amino acid modified of L124E, A125S, H172R and K228D or combinations thereof；

L1 is comprising selected from the amino acid modified of S121K, V133G and S176R or combinations thereof；

H2 is comprising selected from the amino acid modified of L124R, A125R and H172T or combinations thereof；And

L2 is comprising selected from the amino acid modified of V133G, N137K, S174R, S176D and T178D or combinations thereof.

22. constructs according to claim 21, wherein：

H1 includes amino acid modified L124E and K228D；

L1 includes amino acid modified S121K, V133G and S176R；

H2 includes amino acid modified L124R and A125R；And

L2 includes amino acid modified V133G and S176D.

23. constructs according to claim 21, wherein：

H1 includes amino acid modified L124E and H172R；

L1 includes amino acid modified V133G and S176R；

H2 includes amino acid modified L124R and H172T；And

L2 includes amino acid modified V133G, S174R and S176D.

24. constructs according to claim 1, wherein

I () H1 and/or H2 is amino acid modified comprising at least one or a group at L124, A139 and V190, and

(ii) L1 and/or L2 is amino acid modified comprising at least one or a group at F116, V133, L135 and S176.

25. constructs according to claim 24, wherein

I () H1 and/or H2 includes at least one or one group of amino acid selected from L124E, L124R, A139W, A139G and V190A Modification；And

(ii) L1 and/or L2 includes at least one or a group selected from F116A, V133G, L135V, L135W, S176R and S176D It is amino acid modified.

26. constructs according to claim 25, wherein

H1 is comprising selected from the amino acid modified of L124E and A139W or combinations thereof；

L1 is comprising selected from the amino acid modified of F116A, V133G, L135V and S176R or combinations thereof；

H2 is comprising selected from the amino acid modified of L124R, A139G and V190A or combinations thereof；And

L2 is comprising selected from the amino acid modified of V133G, L135W and S176D or combinations thereof.

27. constructs according to claim 26, wherein：

H1 includes amino acid modified L124E and A139W；

L1 includes amino acid modified F116A, V133G, L135V and S176R；

H2 includes amino acid modified L124R, A139G and V190A；And

L2 includes amino acid modified V133G, L135W and S176D.

28. constructs according to claim 1, wherein

I () H1 and/or H2 is amino acid modified comprising at least one or a group at Q39, L45, K145, H172, Q179 and S186, And

(ii) L1 and/or L2 includes at least one or one group of amino at Q38, P44, Q124, S131, Q160, T180 and C214 Acid modification.

29. constructs according to claim 28, wherein

I () H1 and/or H2 includes at least one or selected from Q39E, Q39R, L45P, K145T, H172R, Q179E and S186R Group is amino acid modified；And

(ii) L1 and/or L2 is included selected from Q38R, Q38E, P44F, Q124E, S131K, Q160E, T180E and C214S at least One or one group amino acid modified.

30. constructs according to claim 29, wherein

H1 is comprising selected from the amino acid modified of Q39E, L45P, K145T, H172R and Q179E or combinations thereof；

L1 is comprising selected from the amino acid modified of Q38R, P44F and S131K or combinations thereof；

H2 is comprising selected from the amino acid modified of Q39R, H172R and S186R or combinations thereof；And

L2 is comprising selected from the amino acid modified of Q38E, Q124E, Q160E, T180E and C214S or combinations thereof.

31. constructs according to claim 30, wherein：

H1 includes amino acid modified Q39E, K145T and Q179E；

L1 includes amino acid modified Q38R and S131K；

H2 includes amino acid modified Q39R and S186R；And

L2 includes amino acid modified Q38E, Q124E, Q160E and T180E.

32. constructs according to claim 30, wherein：

H1 includes amino acid modified L45P, K145T, H172R and Q179E；

L1 includes amino acid modified P44F and S131K；

H2 includes amino acid modified H172R and S186R；And

L2 includes amino acid modified Q124E, Q160E and T180E.

33. constructs according to claim 1, wherein

I () H1 and/or H2 is amino acid modified comprising at least one or a group at A139, L143, K145, Q179 and V190, and And

(ii) L1 and/or L2 is repaiied comprising at least one or one group of amino acid at F116, Q124, L135, Q160, T178 and T180 Decorations.

34. constructs according to claim 33, wherein

I () H1 and/or H2 includes at least one selected from A139W, A139G, L143E, K145T, Q179E, Q179K and V190A Or one group amino acid modified；And

(ii) L1 and/or L2 is included selected from F116A, Q124R, Q124E, L135V, L135W, Q160E, T178R and T180E extremely Few one or one group amino acid modified.

35. constructs according to claim 34, wherein

H1 is comprising selected from the amino acid modified of A139W, L143E, K145T and Q179E or combinations thereof；

L1 is comprising selected from the amino acid modified of F116A, Q124R, L135V and T178R or combinations thereof；

H2 is comprising selected from the amino acid modified of A139G, Q179K and V190A or combinations thereof；And

L2 is comprising selected from the amino acid modified of Q124E, L135W, Q160E and T180E or combinations thereof.

36. constructs according to claim 35, wherein：

H1 includes amino acid modified A139W, L143E, K145T and Q179E；

L1 includes amino acid modified F116A, Q124R, L135V and T178R；

H2 includes amino acid modified Q179K；And

L2 includes amino acid modified Q124E, L135W, Q160E and T180E.

37. constructs according to claim 1, wherein

I () H1 and/or H2 is repaiied comprising at least one or one group of amino acid at Q39, L143, K145, D146, H172 and Q179 Decorations, and

(ii) L1 and/or L2 is amino acid modified comprising at least one or a group at Q38, Q124, Q160, T178 and T180.

38. constructs according to claim 37, wherein

I () H1 and/or H2 is included selected from Q39E, Q39R, L143E, K145T, D146G, H172R, Q179E and Q179K at least One or one group amino acid modified；And

(ii) L1 and/or L2 is included selected from Q38R, Q38E, Q124R, Q124E, Q160K, Q160E, T178R and T180E at least One or one group amino acid modified.

39. constructs according to claim 38, wherein

H1 is comprising selected from the amino acid modified of Q39E, L143E, K145T, H172R and Q179E or combinations thereof；

L1 is comprising selected from the amino acid modified of Q38R, Q124R, Q160K and T178R or combinations thereof；

H2 is comprising selected from the amino acid modified of Q39R, H172R and Q179K or combinations thereof；And

L2 is comprising selected from the amino acid modified of Q38E, Q124E, D146G, Q160E and T180E or combinations thereof.

40. constructs according to claim 39, wherein：

H1 includes amino acid modified Q39E, L143E, K145T and Q179E；

L1 includes amino acid modified Q38R, Q124R, Q160K and T178R；

H2 includes amino acid modified Q39R, H172R and Q179K；And

L2 includes amino acid modified Q38E, Q124E, Q160E and T180E.

41. constructs according to claim 1, wherein

I () H1 and/or H2 is repaiied comprising at least one or one group of amino acid at L45, L143, K145, D146, H172 and Q179 Decorations, and

(ii) L1 and/or L2 includes at least one at Q38, P44, Q124, N137, Q160, S174, T178, T180 and C214 Or one group amino acid modified.

42. constructs according to claim 41, wherein

I () H1 and/or H2 is included selected from L45P, L143E, K145T, D146G, H172R, H172T, Q179E and Q179K at least One or one group amino acid modified；And

(ii) L1 and/or L2 comprising selected from Q38E, P44F, Q124R, Q124E, N137K, Q160K, Q160E, S174R, T178R, At least one or one group of T180E and C214S is amino acid modified.

43. constructs according to claim 42, wherein

H1 is comprising selected from the amino acid modified of L45P, L143E, K145T, H172R and Q179E or combinations thereof；

L1 is comprising selected from the amino acid modified of P44F, Q124R, Q160K and T178R or combinations thereof；

H2 is comprising selected from the amino acid modified of D146G, H172R, H172T and Q179K or combinations thereof；And

L2 is repaiied comprising the amino acid selected from Q38E, Q124E, N137K, Q160E, S174R, T180E and C214S or combinations thereof Decorations.

44. constructs according to claim 43, wherein：

H1 includes amino acid modified L45P, L143E and K145T；

L1 includes amino acid modified P44F, Q124R, Q160K and T178R；

H2 includes amino acid modified D146G and Q179K；And

L2 includes amino acid modified Q38E, Q124E, Q160E and T180E.

45. constructs according to claim 43, wherein：

H1 includes amino acid modified L143E, K145T and H172R；

L1 includes amino acid modified Q124R, Q160K and T178R；

H2 includes amino acid modified H172T and Q179K；And

L2 includes amino acid modified Q124E, Q160E, N137K, S174R and T180E.

46. constructs according to claim 1, wherein

I () H1 and/or H2 is amino acid modified comprising at least one or a group at L124, L143, K145 and Q179, and

(ii) L1 and/or L2 is repaiied comprising at least one or one group of amino acid at Q124, S131, V133, S176, T178 and T180 Decorations.

47. constructs according to claim 46, wherein

I () H1 and/or H2 includes at least one selected from L124W, L124A, L143E, L143F, K145T, Q179E and Q179K Or one group amino acid modified；And

(ii) L1 and/or L2 comprising selected from Q124R, Q124K, Q124E, S131K, V133A, V133W, S176T, T178R, At least one or one group of T178L, T178E and T180E is amino acid modified.

48. constructs according to claim 47, wherein

H1 is comprising selected from the amino acid modified of L124W, L143E, K145T and Q179E or combinations thereof；

L1 includes the amino acid selected from Q124R, Q124K, S131K, V133A, S176T, T178R and T178L or combinations thereof Modification；

H2 is comprising selected from the amino acid modified of L124A, L143F and Q179K or combinations thereof；And

L2 is comprising selected from the amino acid modified of Q124E, V133W, S176T, T178L, T178E and T180E or combinations thereof.

49. constructs according to claim 48, wherein：

H1 includes amino acid modified L124W, L143E, K145T and Q179E；

L1 includes amino acid modified Q124R, V133A, S176T and T178R；

H2 includes amino acid modified L124A, L143F and Q179K；And

L2 includes amino acid modified Q124E, V133W, S176T, T178L and T180E.

50. constructs according to claim 1, wherein

I () H1 and/or H2 is amino acid modified comprising at least one or a group at A139, L143, K145, Q179 and S186, and And

(ii) L1 and/or L2 is repaiied comprising at least one or one group of amino acid at F116, Q124, V133, Q160, T178 and T180 Decorations.

51. constructs according to claim 50, wherein

(i) H1 and/or H2 comprising selected from A139C, L143E, L143D, L143R, L143K, K145T, Q179E, Q179D, At least one or one group of Q179R, Q179K, S186K, S186R is amino acid modified；And

(ii) L1 and/or L2 comprising selected from F116C, Q124R, Q124K, Q124E, V133E, V133D, Q160K, Q160E, At least one or one group of T178R, T178K, T178E and T180E is amino acid modified.

52. constructs according to claim 51, wherein

H1 is comprising selected from the amino acid modified of A139C, L143E, L143D, K145T, Q179E and Q179D or combinations thereof；

L1 is comprising selected from the amino acid modified of F116C, Q124R, Q124K, Q160K, T178R and T178K or combinations thereof；

H2 is comprising selected from the amino acid modified of L143R, L143K, Q179R, Q179K, S186K and S186R or combinations thereof；And And

L2 is comprising selected from the amino acid modified of Q124E, V133E, V133D, Q160E, T178E and T180E or combinations thereof.

53. constructs according to claim 52, wherein：

H1 includes amino acid modified A139C, L143E, K145T and Q179E；

L1 includes amino acid modified F116C, Q124R and T178R；

H2 includes amino acid modified Q179K；And

L2 includes amino acid modified Q124E, Q160E and T180E.

54. constructs according to claim 52, wherein：

H1 includes amino acid modified L143E, K145T and Q179E；

L1 includes amino acid modified Q124R and T178R；

H2 includes amino acid modified S186K；And

L2 includes amino acid modified Q124E, Q160E and T178E.

55. constructs according to claim 52, wherein：

H1 includes amino acid modified L143E, K145T and Q179E；

L1 includes amino acid modified Q124R and T178R；

H2 includes amino acid modified L143R；And

L2 includes amino acid modified Q124E and V133E.

56. constructs according to claim 1, wherein

I () H1 and/or H2 includes at least one or one group of amino at L124, L143, K145, D146, Q179, S186 and S188 Acid modification, and

57. constructs according to claim 56, wherein

(i) H1 and/or H2 comprising selected from L124A, L143A, L143R, L143E, L143K, K145T, D146G, Q179R, At least one or one group at Q179E, Q179K, S186R, S186K and S188L is amino acid modified；And

(ii) L1 and/or L2 comprising selected from Q124R, Q124E, S131E, S131T, V133Y, V133W, V133E, V133D, At least one or one group of amino acid of Q160E, Q160K, Q160M, S176L, T178R, T178E, T178F, T178Y and T180E Modification.

58. constructs according to claim 57, wherein

H1 is comprising selected from the amino acid modified of L143E, K145T, Q179E and S188L or combinations thereof；

L1 is comprising selected from the amino acid modified of Q124R, Q160K and T178R or combinations thereof；

H2 comprising selected from L124A, L143A, L143R, L143K, D146G, Q179R, Q179K, S186R and S186K or they That what is combined is amino acid modified；And

L2 comprising selected from Q124E, S131E, S131T, V133Y, V133W, V133E, V133D, Q160E, Q160M, S176L, T178E, T178F, T178Y and T180E or combinations thereof it is amino acid modified.

59. constructs according to claim 58, wherein：

H1 includes amino acid modified L143E, K145T, Q179E and S188L；

L1 includes amino acid modified Q124R and T178R；

H2 includes amino acid modified S186K；And

L2 includes amino acid modified Q124E, S176L and T180E.

60. constructs according to claim 58, wherein：

H1 includes amino acid modified L143E, K145T, Q179E and S188L；

L1 includes amino acid modified Q124R and T178R；

H2 includes amino acid modified S186K；And

L2 includes amino acid modified Q124E, S131T, T178Y and T180E.

61. constructs according to claim 58, wherein：

H1 includes amino acid modified L143E and K145T；

L1 includes amino acid modified Q124R, Q160K and T178R；

H2 includes amino acid modified S186K；And

L2 includes amino acid modified S131E.

62. constructs according to claim 58, wherein：

H1 includes amino acid modified L143E and K145T；

L1 includes amino acid modified Q124R；

H2 includes amino acid modified L143R；And

L2 includes amino acid modified Q124E and V133E.

63. constructs according to claim 1, wherein

I () H1 is amino acid modified comprising at least one or a group at F122 and C233, and

(ii) L1 is amino acid modified comprising at least one or a group at Q124 and C214.

64. constructs according to claim 63, wherein

I () H1 is amino acid modified comprising at least one or a group selected from F122C and C233S；And

(ii) L1 is amino acid modified comprising at least one or a group selected from Q124C and C214S.

65. constructs according to claim 64, wherein

H1 is comprising selected from the amino acid modified of F122C and C233S or combinations thereof；

L1 is comprising selected from the amino acid modified of Q124C and C214S or combinations thereof；

H2 includes wild type or not modified amino acid sequence；And

L2 includes wild type or not modified amino acid sequence.

66. constructs according to claim 65, wherein：

H1 includes amino acid modified F122C and C233S；

L1 includes amino acid modified Q124C and C214S；

H2 includes wild type or not modified amino acid sequence；And

L2 includes wild type or not modified amino acid sequence.

67. according to construct in any one of the preceding claims wherein, wherein when H1, H2, L1 and L2 are dynamic in cell or lactation When being co-expressed in thing cell, or when H1, H2, L1 and L2 are co-expressed in Cell free expression system, or as H1, H2, L1 and L2 Symbiosis into when, or when H1, H2, L1 and L2 by the symbiosis of redox generation method into when, H1 preferentially matches somebody with somebody with L1 compared with L2 It is right, and H2 is preferentially matched with L2 compared with L1.

68. include according to construct in any one of the preceding claims wherein, wherein at least one of H1, H2, L1 and L2 At least one of VH and/or VL domains is amino acid modified, and/or at least one amino acid of CH1 and/or CL domains is repaiied Decorations so that H1 is preferentially matched with L1 compared with L2, and/or H2 is preferentially matched with L2 compared with L1.

69. according to construct in any one of the preceding claims wherein, if wherein H1 is included in the CH1 domains extremely Few one amino acid modified, then at least one of L1 and L2 are amino acid modified comprising at least one of the CL domains； And/or if H1 is amino acid modified comprising at least one of the VH domains, then at least one of L1 and L2 include institute State at least one of VL domains amino acid modified；If or wherein H2 is comprising at least one of CH1 domains ammonia Base acid modification, then at least one of L1 and L2 are amino acid modified comprising at least one of the CL domains；And/or if H2 is amino acid modified comprising at least one of the VH domains, then at least one of L1 and L2 include the VL domains At least one of it is amino acid modified.

70. include the Fab areas according to construct in any one of the preceding claims wherein, wherein H1, L1, H2 and/or L2 In at least 1,2,3,4,5,6,7,8,9 or 10 amino acid mutations.

71. include according to construct in any one of the preceding claims wherein, wherein at least one of H1, H2, L1 and L2 At least 2,3,4,5,6,7,8,9 or 10 amino acid of at least one constant domain and/or at least one variable domains are repaiied Decorations.

72. according to construct in any one of the preceding claims wherein, wherein when in both L1 and L2 with H1 and H2 at least One coexpression when, H1-L1 and H2-L2 heterodimer centerings described at least one opposing pairs it is respective corresponding with described H1-L2 or H2-L1 heterodimers to ratio more than 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%th, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%th, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%th, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and wherein described Jing repaiies The opposing pairs of the H1-L1 or H2-L2 heterodimers pair of decorations are more than without the described at least one amino acid modified correspondence The respective opposing pairs observed of H1-L1 or H2-L2 heterodimer centerings.

73. according to construct in any one of the preceding claims wherein, wherein in first and second heterodimer The heat endurance at least one Fab areas is described right without described at least one amino acid modified or amino acid modified group Within about 0,1,2 or 3 DEG C of the Tm of the heterodimer answered.

74. according to construct in any one of the preceding claims wherein, the antigen that wherein each heterodimer is combined to it Affinity each not modified heterodimer to about the 1 of the affinity of identical antigen, 2,3,4,5,6,7, 8th, within 9,10,20,25,30,35,40,45 or 50 times, as determined by surface plasmon resonance (SPR) or FACS.

75. according to construct in any one of the preceding claims wherein, wherein

At least one of H1 and L1 include domain as at least one：Its include it is at least one amino acid modified, with L2 Compare and produce bigger amino acid spatial complementary when H1 and L1 is matched；Or

Wherein at least one of H2 and L2 include domain as at least one：Its include it is at least one amino acid modified, Bigger amino acid spatial complementary is produced compared with L1 when H2 and L2 pairings；Or

Wherein at least one of H1 and L1 include domain as at least one：Its include it is at least one amino acid modified, Electrostatic complementarities compared with L2 when H1 and L1 is matched between the bigger Charged acids of generation；Or

Wherein at least one of H2 and L2 include domain as at least one：Its include it is at least one amino acid modified, Electrostatic complementarities compared with L1 when H2 and L2 is matched between the bigger Charged acids of generation；Or

Wherein at least one of H1 and L1 include domain as at least one：Its include it is at least one amino acid modified, Bigger amino acid space and electrostatic complementarities are produced when H1 and L1 are matched compared with L2；Or

Wherein at least one of H2 and L2 include domain as at least one：Its include it is at least one amino acid modified, Bigger amino acid space and electrostatic complementarities are produced when H2 and L2 are matched compared with L1；Or

Wherein at least one of H1 and L1 include domain as at least one, its include it is at least one amino acid modified, Covalent bond is produced between H1 and L1；Or

Wherein at least one of H2 and L2 include domain as at least one, its include it is at least one amino acid modified, Covalent bond is produced between H2 and L2.

76. according to construct in any one of the preceding claims wherein, wherein H1, H2, L1 and L2 comprising one group selected from table 5 to It is amino acid modified that unique identifier design shown in table 6, table 15 to table 27 or table 28a to table 28c collects.

77. according to construct in any one of the preceding claims wherein, wherein the construct also includes Fc, the Fc is included First CH3 sequences and the 2nd CH3 sequences, wherein the first CH3 sequences by or be not connected to by one or more joints First heterodimer, and the 2nd CH3 sequences by or by one or more joints be not connected to described Two heterodimers.

78. constructs according to claim 77, wherein the Fc is people Fc, human IgG1 Fc, people IgA Fc, human IgG Fc, people IgD Fc, people IgE Fc, people IgM Fc, human IgG2 Fc, human IgG 3Fc or human IgG 4Fc.

79. constructs according to claim 77 or 78, wherein the Fc is heterodimer Fc.

80. constructs according to any one of claim 77 to 79, wherein the Fc is included in the CH3 sequences extremely One or more modifications in few one.

81. constructs according to any one of claim 77 to 80, wherein the dimerization CH3 sequences have such as DSF Determined about 68 DEG C, 69 DEG C, 70 DEG C, 71 DEG C, 72 DEG C, 73 DEG C, 74 DEG C, 75 DEG C, 76 DEG C, 77 DEG C, 77.5 DEG C, 78 DEG C, 79 DEG C, 80 DEG C, 81 DEG C, 82 DEG C, 83 DEG C, 84 DEG C or 85 DEG C or higher of melt temperature (Tm).

82. constructs according to any one of claim 77 to 81, wherein in the preparation, the Fc is with greater than about 75%th, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%th, the heterodimer that 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity is formed；Or its In in expression or when expressing via individual cells, the Fc be to be greater than about 75%, 76%, 77%, 78%, 79%, 80%th, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%th, the heterodimer that 96%, 97%, 98% or 99% purity is formed.

83. constructs according to any one of claim 77 to 82, wherein the Fc is included in the CH3 sequences extremely One or more modifications in few one, the modification contributes to forming stability different equivalent to wild-type homologous dimeric Fc Source dimeric Fc.

84. constructs according to any one of claim 78 to 84, wherein the Fc is included：

I) there is the modification T366L_K392M_ in the modification L351Y_F405A_Y407V and the 2nd Fc polypeptides in a Fc polypeptides The heterodimer IgG1Fc of T394W；

V) there is the modification in the modification T350V_L351Y_S400E_F405A_Y407V and the 2nd Fc polypeptides in a Fc polypeptides The heterodimer IgG1Fc of T350V_T366L_N390R_K392M_T394W.

85. constructs according to any one of claim 77 to 84, wherein the Fc also includes at least one CH2 sequences Row.

86. constructs according to claim 85, wherein the CH2 sequences of the Fc include one or more modifications.

87. constructs according to any one of claim 77 to 86, wherein the Fc comprising one or more modifications with Contribute to the selective binding of Fc- γ acceptors.

88. constructs according to claim 77 to 87 is in any one, wherein the Fc passes through one or more joint idols The heterodimer is linked to, or wherein described Fc is coupled to H1 and H2 by one or more joints.

89. constructs according to claim 88, wherein one or more of joints are one or more peptide linkers.

90. constructs according to claim 88, wherein one or more of joints include one or more antibody hinges Sequence.

91. constructs according to claim 88, wherein one or more of joints include one or more IgG1 hinges Sequence.

92. constructs according to any one of claim 88 to 91, wherein one or more of joints include one Or multiple modifications.

93. constructs according to claim 92, wherein one or more of modify the selection for contributing to Fc- γ acceptors Property combine.

94. according to construct in any one of the preceding claims wherein, wherein described at least one it is amino acid modified be at least One amino acid mutation, or it is wherein described at least one it is amino acid modified be at least one amino acid replacement.

95. according to construct in any one of the preceding claims wherein, the sequence of each wherein in H1, H2, L1 and L2 From human sequence.

96. according to construct in any one of the preceding claims wherein, wherein the construct is polyspecific or double spies The opposite sex.

97. according to construct in any one of the preceding claims wherein, wherein the construct is multivalence or divalence.

A kind of 98. detached polynucleotides or detached polynucleotides group, it includes at least one coding will according to aforementioned right Construct sequence any one of asking.

The 99. detached polynucleotides according to claim 98 or detached polynucleotides group, wherein the polynucleotides Or polynucleotides group is cDNA.

A kind of 100. carriers or vehicle group, it is included in the polynucleotides according to claim 98 or 99 or polynucleotides group One or more.

101. carriers or vehicle group according to claim 100, it is selected from plasmid, polycistronic vector, viral vector, non- Episomal mammalian vectors, expression vector and recombinant expression carrier.

A kind of 102. detached cells, it includes the polynucleotides according to claim 98 or 99 or polynucleotides group, or Carrier or vehicle group according to claim 100 or 101.

The 103. detached cells according to claim 102, wherein the cell is yeast cells, bacterial cell, hybridization Knurl, Chinese hamster ovary (CHO) cell or HEK293 cells.

104. a kind of pharmaceutical compositions, comprising according to construct in any one of the preceding claims wherein and pharmaceutically acceptable Carrier.

105. pharmaceutical compositions according to claim 104, also comprising one or more selected from buffer solution, antioxidant, The material of low-molecular-weight molecule, medicine, protein, amino acid, carbohydrate, lipid, chelating agent, stabilizer and excipient.

106. constructs according to claim 1 to 96 or the pharmaceutical composition according to claim 104 or 105 are used In the disease or illness treated in experimenter or for the purposes in medicine manufacture.

A kind of 107. methods for the treatment of with disease or the experimenter of illness, methods described includes applying basis to the experimenter Construct any one of claim 1 to 96 or the pharmaceutical composition according to claim 104 or 105.

A kind of 108. methods that the construct according to any one of claim 1 to 96 is obtained from host cell cultures, The method comprising the steps of：

A () obtains the host cell cultures comprising at least one host cell, the host cell is compiled comprising one or more The nucleotide sequence of the code construct；And

B () reclaims the construct from the host cell cultures.

A kind of method of construct of 109. acquisitions according to any one of claim 1 to 96, methods described includes following Step：

A () obtains H1, L1, H2 or L2；

B () allows the H1 compared with L2 preferentially to match with L1, and H2 is preferentially matched with L2 compared with L1；And

C () obtains the construct.

A kind of 110. methods of the construct prepared according to any one of claim 1 to 96, methods described includes：

A. the polynucleotides or polynucleotides group of at least one construct of coding are obtained；

B. determine for the optimal of each in being introduced into the polynucleotides of at least one host cell or polynucleotides group Ratio, wherein the best ratio is different with the H1-L2 and H2-L1 of the mispairing formed when H1, L1, H2 or L2 are expressed by assessing Source dimer determines to the amount for comparing H1-L1 the and H2-L2 heterodimers pair formed when H1, L1, H2 or L2 are expressed；

C. preferred best ratio is selected, wherein with the polynucleotides of the preferred best ratio or polynucleotides group At least one host cell of transfection causes the construct to be expressed；

D. at least one host cell is transfected with the polynucleotides or polynucleotides group of the best ratio；And

E. cultivate at least one host cell to express the construct.

111. methods according to claim 110, wherein selecting the best ratio by transient transfection system transfer Contaminate to assess.

112. methods according to claim 110 or 111, wherein with the polynucleotides of the preferred best ratio Or polynucleotides group transfects at least one host cell and causes the optimum expression of the construct.

113. methods according to any one of claim 110 to 112, wherein the construct includes Fc, the Fc bags Containing at least two CH3 sequences, wherein the Fc by or by one or more joints be not coupled to first heterodimeric Body and second heterodimer.

114. methods according to claim 113, wherein the Fc is heterodimer, optionally comprising one or more It is amino acid modified.

A kind of 115. computer-readable recording mediums, the media storage：

Data set comprising the data for representing the first heterodimer and the complemented mutant in the second heterodimer, described first Heterodimer includes the first heavy chain immunoglobulin peptide sequence (H1) and the first light chain immunoglobulin peptide sequence (L1)； And second heterodimer includes the second heavy chain immunoglobulin peptide sequence (H2) and the second light chain immunoglobulin Peptide sequence (L2), each self-contained at least heavy-chain variable domains (VH domains) of wherein H1 and H2 and heavy chain constant domain (CH1 domains)；The each self-contained at least light variable domains (VL domains) of wherein L1 and L2 and light chain constant domain (CL Domain), and the data set of wherein described complemented mutant is comprising expression table 4 to table 6, table 15 to table 27, table 28a to table 28c Those mutation listed or the data of the subset of those mutation.

A kind of 116. methods for preparing bispecific antigen-binding polypeptides construct, the bispecific construct is different comprising first Source dimer and the second heterodimer, first heterodimer is included from the first monospecific antigen-binding polypeptides First heavy chain immunoglobulin peptide sequence (H1) and the first light chain immunoglobulin peptide sequence (L1)；And described second is different Source dimer includes the second heavy chain immunoglobulin peptide sequence (H2) from the second monospecific antigen-binding polypeptides and the Two light chain immunoglobulin peptide sequences (L2), each self-contained at least heavy-chain variable domains (VH domains) of wherein H1 and H2 and Heavy chain constant domain (CH1 domains)；Each self-contained at least light variable domains (VL domains) of wherein L1 and L2 and light Chain constant domain (CL domains), methods described includes：

A. one or more complemented mutants of the data set according to claim 115 are introduced into first heterodimer And/or second heterodimer；And

B. first heterodimer and second heterodimer is made to be co-expressed at least one host cell, with life Into the expression product comprising the bispecific construct.

117. methods according to claim 116, it also includes determining the expression product relative to other polypeptide products Described in bispecific construct amount, to select preferred complemented mutant subset.

118. methods according to claim 116 or 117, wherein compared with other polypeptide products, the bispecific structure Build body and generated with the purity more than 70%.

119. methods according to any one of claim 116 to 118, it is also included other amino acid modified addition At least one of to H1, H2, L1 or L2, to improve the bispecific construct compared to the purity of other polypeptide products Step.

120. methods according to any one of claim 116 to 119, wherein the construct includes Fc, the Fc bags Containing at least two CH3 sequences, wherein the Fc by or by one or more joints be not coupled to first heterodimeric Body and second heterodimer.

121. methods according to claim 120, wherein the Fc is heterodimer, optionally comprising one or more It is amino acid modified.

122. methods according to any one of claim 116 to 121, wherein the antigen-binding polypeptides are antibody, list Chain monoclonal antibody, Fab or single chain Fab.

A kind of 123. sides of the detached antigen-binding polypeptides construct prepared according to any one of claim 1 to 96 Method, methods described is included in expression H1, H2, L1 and L2 at least one expression system, to generate expression product；Characterize the table Up to product；And select the expression product wherein more than 90% to be the described of the detached antigen-binding polypeptides construct Expression system.

124. constructs according to claim 1, wherein the amino acid modified unique mark selected from corresponding to table 35a The SMCA designs of symbol

9561-9095_1,9561-9095_2, wherein H1 include L124W, L143E, K145T and Q179E, L1 comprising Q124R, V133A, S176T and T178R, H2 includes L124A, L143F and Q179K, and L2 includes Q124E, V133W, S176T, T178L And T180E；

9121-9373_1,9121-9373_2, wherein H1 comprising L124E and H172T, L1 comprising V133G, N137K, S174R and S176R, H2 include L124R and H172R, and L2 includes V133G, S176D and T178D；

9116-9349_1,9116-9349_2, wherein H1 comprising L124E and A139W, L1 comprising F116A, V133G, L135V and S176R, H2 include L124R, A139G and V190A, and L2 includes V133G, L135W and S176D；

9134-9521_1,9134-9521_2, wherein H1 include L124E, K145T and Q179E, L1 comprising S131K, V133G and S176R, H2 include L124R and S186R, and L2 includes V133G, S176D and T178D；

9286-9402_1,9286-9402_2, wherein H1 include L124E, L143E and K145T, L1 comprising Q124K, V133G and S176R, H2 include L124R and Q179K, and L2 includes V133G, S176D and T178E；

9667-9830_1,9667-9830_2, wherein H1 include L143E, K145T and Q179E, and L1 includes Q124R and T178R, H2 includes S186K, and L2 includes Q124E, Q160E and T178E；

9696-9848_1,9696-9848_2, wherein H1 include L143E, K145T, Q179E and S188L, L1 comprising Q124R and T178R, H2 include S186K, and L2 includes Q124E, S176L and T180E；

9060-9756_1,9060-9756_2, wherein H1 include A139W, L143E, K145T and Q179E, L1 comprising F116A, Q124R, L135V and T178R, H2 includes Q179K, and L2 includes Q124E, L135W, Q160E and T180E；

9682-9740_1,9682-9740_2, wherein H1 include L143E, K145T and Q179E, and L1 includes Q124R and T178R, H2 includes L143R, and L2 includes Q124E and V133E；

9049-9759_1,9049-9759_2, wherein H1 include A139C, L143E, K145T and Q179E, L1 comprising F116C, Q124R and T178R, H2 include Q179K, and L2 includes Q124E, Q160E and T180E；And

9820-9823_1,9820-9823_2, wherein H1 include Q39E, L143E, K145T and Q179E, L1 comprising Q38R, Q124R, Q160K and T178R, H2 includes Q39R, H172R and Q179K, and L2 includes Q38E, Q124E, Q160E and T180E.

125. constructs according to claim 1, wherein the amino acid modified unique mark selected from corresponding to table 35b The SMCA designs of symbol

9327-6054_1, wherein H1 include L124R, and L2 comprising L124E and L143F, L1 comprising V133G and S176R, H2 Comprising V133G, S176D and T178D；

9815-9825_1,9815-9825_2, wherein H1 include Q38R, V133G and S176R, H2 comprising Q39E and L124E, L1 Comprising Q39R and L124R, and L2 includes Q38E, V133G and S176D；

9587-9735_l, 9587-9735_2, wherein H1 include Q124R comprising L143E and K145T, L1, and H2 includes L143R, and And L2 includes Q124E and VI33E；

3522_1,3522_2, wherein H1 include L45P, L143E and K145T, and L1 includes P44F, Q124R, Q160K and T178R, H2 includes D146G and Q179K, and L2 includes Q124E, Q160E and T180E；And

3519_1,3519_2, wherein H1 include L45P, K145T, H172R and Q179E, and L1 is included comprising P44F and S131K, H2 H172R and S186R, and L2 includes Q124E, Q160E and T180E.

126. constructs according to claim 1, wherein described amino acid modified selected from corresponding to unique identifier SMCA is designed

9060-9756, wherein H1 include A139W, L143E, K145T and Q179E, L1 comprising F116A, Q124R, L135V and T178R, H2 include Q179K, and L2 includes Q124E, L135W, Q160E and T180E；

9820-9823, wherein H1 include Q39E, L143E, K145T and Q179E, L1 comprising Q38R, Q124R, Q160K and T178R, H2 include Q39R, H172R and Q179K, and L2 includes Q38E, Q124E, Q160E and T180E,

3519, wherein H1 include L45P, K145T, H172R and Q179E, L1 comprising P44F and S131K, H2 comprising H172R and S186R, and L2 includes Q124E, Q160E and T180E,

9049-9759, wherein H1 include A139C, L143E, K145T and Q179E, and L1 includes F116C, Q124R and T178R, H2 Comprising Q179K, and L2 includes Q124E, Q160E and T180E；

3522, wherein H1 include L45P, L143E and K145T, and L1 includes P44F, Q124R, Q160K and T178R, and H2 is included D146G and Q179K, and L2 includes Q124E, Q160E and T180E；

9696-9848, wherein H1 include L143E, K145T, Q179E and S188L, and L1 is included comprising Q124R and T178R, H2 S186K, and L2 includes Q124E, S176L and T180E；

9692-9846, wherein H1 include L143E, K145T, Q179E and S188L, and L1 is included comprising Q124R and T178R, H2 S186K, and L2 includes Q124E, S131T, T178Y and T180E；

9986-9978, wherein H1 include Q124R, Q160K and T178R comprising L143E and K145T, L1, and H2 includes S186K, and And L2 includes S131E, and

9667-9830, wherein H1 include L143E, K145T and Q179E, and L1 includes S186K comprising Q124R and T178R, H2, and And L2 includes Q124E, Q160E and T178E.

127. constructs according to claim 1, wherein described amino acid modified selected from corresponding to unique identifier SMCA is designed

9587-9735, wherein H1 include Q124R comprising L143E and K145T, L1, and H2 includes L143R, and L2 includes Q124E And V133E；

9561-9095, wherein H1 include L124W, L143E, K145T and Q179E, L1 comprising Q124R, V133A, S176T and T178R, H2 include L124A, L143F and Q179K, and L2 includes Q124E, V133W, S176T, T178L and T180E；

9611-9077, wherein H1 include L143E, K145T and H172R, and L1 includes Q124R, Q160K and T178R, and H2 is included H172T and Q179K, and L2 includes Q124E, N137K, Q160E, S174R and T180E；

9168-9342, wherein H1 include S121K, V133G and S176R comprising L124E and K228D, L1, H2 comprising L124R and A125R, and L2 includes V133G and S176D；

9164-9555, wherein H1 include L124E, K145T and Q179E, and L1 includes S131R, V133G and S176R, and H2 is included L124R and S186R, and L2 includes V133G, S176D and T180E；

9279-9518, wherein H1 include L124E, L143E and K145T, and L1 includes Q124K, V133G and S176R, and H2 is included L124R and S186R, and L2 includes V133G, S176D and T178D；

9290-9432, wherein H1 include L124E, L143E and K145T, and L1 includes Q124K, V133G and S176R, and H2 is included L124R and Q179K, and L2 includes V133G, S176D and T180E；

9142-9414, wherein H1 include L124E, K145T and Q179E, and L1 includes S131K, V133G and S176R, and H2 is included L124R and Q179K, and L2 includes V133G, S176D and T178E；

9121-9373, wherein H1 include V133G, N137K, S174R and S176R comprising L124E and H172T, L1, and H2 is included L124R and H172R, and L2 includes V133G, S176D and T178D；

9066-9335, wherein H1 include Q124C, V133G and S176R comprising F122C and L124E, L1, and H2 includes L124R, and And L2 includes V133G and S176D；

9820-9823, wherein H1 include Q39E, L143E, K145T and Q179E, L1 comprising Q38R, Q124R, Q160K and T178R, H2 include Q39R, H172R and Q179K, and L2 includes Q38E, Q124E, Q160E and T180E；

9814-9828, wherein H1 include Q39E, K145T and Q179E, L1 comprising Q38R and S131K, H2 comprising Q39R and S186R, and L2 includes Q38E, Q124E, Q160E and T180E；

9667-9830, wherein H1 include L143E, K145T and Q179E, and L1 includes S186K comprising Q124R and T178R, H2, and And L2 includes Q124E, Q160E and T178E；

9986-9978, wherein H1 include Q124R, Q160K and T178R comprising L143E and K145T, L1, and H2 includes S186K, and And L2 includes S131E；

3522, wherein H1 include L45P, L143E and K145T, and L1 includes P44F, Q124R, Q160K and T178R, and H2 is included D146G and Q179K, and L2 includes Q124E, Q160E and T180E, and

3519, wherein H1 include L45P, K145T, H172R and Q179E, L1 comprising P44F and S131K, H2 comprising H172R and S186R, and L2 includes Q124E, Q160E and T180E.

Claims

2. construct according to claim 1, wherein

3. construct according to claim 2, wherein

4. construct according to claim 3, wherein：

5. construct according to claim 4, wherein：

H1 includes amino acid modified L124E, K145T and Q179E；

L1 includes amino acid modified S131K, V133G and S176R；

H2 includes amino acid modified L124R and S186R；And

L2 includes amino acid modified V133G, S176D and T178D.

6. construct according to claim 1, wherein

7. construct according to claim 6, wherein

8. construct according to claim 7, wherein：

9. construct according to claim 8, wherein：

H1 includes amino acid modified L124E, L143E and K145T；

L1 includes amino acid modified Q124K, V133G and S176R；

H2 includes amino acid modified L124R and Q179K；And

L2 includes amino acid modified V133G, S176D and T178E.

10. construct according to claim 8, wherein：

H1 includes amino acid modified L124E, L143E and K145T；

L1 includes amino acid modified Q124K, V133G and S176R；

H2 includes amino acid modified L124R and S186R；And

L2 includes amino acid modified V133G, S176D and T178D.

11. constructs according to claim 1, wherein

12. constructs according to claim 11, wherein

13. constructs according to claim 12, wherein

14. constructs according to claim 13, wherein：

H1 includes amino acid modified Q39E and L124E；

L1 includes amino acid modified Q38R, V133G and S176R；

H2 includes amino acid modified Q39R and L124R；And

L2 includes amino acid modified Q38E, V133G and S176D.

15. constructs according to claim 13, wherein：

H1 includes amino acid modified L45P and L124E；

L1 includes amino acid modified P44F, V133G and S176R；

H2 includes amino acid modified L124R；And

L2 includes amino acid modified V133G, S176D and T178D.

16. constructs according to claim 13, wherein：

H1 includes amino acid modified L124E and L143F；

L1 includes amino acid modified V133G and S176R；

H2 includes amino acid modified L124R；And

L2 includes amino acid modified V133G, S176D and T178D.

17. constructs according to claim 13, wherein：

H1 includes amino acid modified F122C and L124E；

L1 includes amino acid modified Q124C, V133G and S176R；

H2 includes amino acid modified L124R；And

L2 includes amino acid modified V133G and S176D.

18. constructs according to claim 13, wherein：

H1 includes amino acid modified L124E and H172T；

L1 includes amino acid modified V133G, N137K, S174R and S176R；

H2 includes amino acid modified L124R and H172R；And

L2 includes amino acid modified V133G, S176D and T178D.

19. constructs according to claim 1, wherein

20. constructs according to claim 19, wherein

21. constructs according to claim 20, wherein

22. constructs according to claim 21, wherein：

H1 includes amino acid modified L124E and K228D；

L1 includes amino acid modified S121K, V133G and S176R；

H2 includes amino acid modified L124R and A125R；And

L2 includes amino acid modified V133G and S176D.

23. constructs according to claim 21, wherein：

H1 includes amino acid modified L124E and H172R；

L1 includes amino acid modified V133G and S176R；

H2 includes amino acid modified L124R and H172T；And

L2 includes amino acid modified V133G, S174R and S176D.

24. constructs according to claim 1, wherein

25. constructs according to claim 24, wherein

26. constructs according to claim 25, wherein

27. constructs according to claim 26, wherein：

H1 includes amino acid modified L124E and A139W；

L1 includes amino acid modified F116A, V133G, L135V and S176R；

H2 includes amino acid modified L124R, A139G and V190A；And

L2 includes amino acid modified V133G, L135W and S176D.

28. constructs according to claim 1, wherein

29. constructs according to claim 28, wherein

30. constructs according to claim 29, wherein

31. constructs according to claim 30, wherein：

H1 includes amino acid modified Q39E, K145T and Q179E；

L1 includes amino acid modified Q38R and S131K；

H2 includes amino acid modified Q39R and S186R；And

L2 includes amino acid modified Q38E, Q124E, Q160E and T180E.

32. constructs according to claim 30, wherein：

H1 includes amino acid modified L45P, K145T, H172R and Q179E；

L1 includes amino acid modified P44F and S131K；

H2 includes amino acid modified H172R and S186R；And

L2 includes amino acid modified Q124E, Q160E and T180E.

33. constructs according to claim 1, wherein

34. constructs according to claim 33, wherein

35. constructs according to claim 34, wherein

36. constructs according to claim 35, wherein：

H1 includes amino acid modified A139W, L143E, K145T and Q179E；

L1 includes amino acid modified F116A, Q124R, L135V and T178R；

H2 includes amino acid modified Q179K；And

L2 includes amino acid modified Q124E, L135W, Q160E and T180E.

37. constructs according to claim 1, wherein

38. constructs according to claim 37, wherein

39. constructs according to claim 38, wherein

40. constructs according to claim 39, wherein：

H1 includes amino acid modified Q39E, L143E, K145T and Q179E；

L1 includes amino acid modified Q38R, Q124R, Q160K and T178R；

H2 includes amino acid modified Q39R, H172R and Q179K；And

L2 includes amino acid modified Q38E, Q124E, Q160E and T180E.

41. constructs according to claim 1, wherein

42. constructs according to claim 41, wherein

43. constructs according to claim 42, wherein

44. constructs according to claim 43, wherein：

H1 includes amino acid modified L45P, L143E and K145T；

L1 includes amino acid modified P44F, Q124R, Q160K and T178R；

H2 includes amino acid modified D146G and Q179K；And

L2 includes amino acid modified Q38E, Q124E, Q160E and T180E.

45. constructs according to claim 43, wherein：

H1 includes amino acid modified L143E, K145T and H172R；

L1 includes amino acid modified Q124R, Q160K and T178R；

H2 includes amino acid modified H172T and Q179K；And

L2 includes amino acid modified Q124E, Q160E, N137K, S174R and T180E.

46. constructs according to claim 1, wherein

47. constructs according to claim 46, wherein

48. constructs according to claim 47, wherein

49. constructs according to claim 48, wherein：

H1 includes amino acid modified L124W, L143E, K145T and Q179E；

L1 includes amino acid modified Q124R, V133A, S176T and T178R；

H2 includes amino acid modified L124A, L143F and Q179K；And

L2 includes amino acid modified Q124E, V133W, S176T, T178L and T180E.

50. constructs according to claim 1, wherein

51. constructs according to claim 50, wherein

52. constructs according to claim 51, wherein

53. constructs according to claim 52, wherein：

H1 includes amino acid modified A139C, L143E, K145T and Q179E；

L1 includes amino acid modified F116C, Q124R and T178R；

H2 includes amino acid modified Q179K；And

L2 includes amino acid modified Q124E, Q160E and T180E.

54. constructs according to claim 52, wherein：

H1 includes amino acid modified L143E, K145T and Q179E；

L1 includes amino acid modified Q124R and T178R；

H2 includes amino acid modified S186K；And

L2 includes amino acid modified Q124E, Q160E and T178E.

55. constructs according to claim 52, wherein：

H1 includes amino acid modified L143E, K145T and Q179E；

L1 includes amino acid modified Q124R and T178R；

H2 includes amino acid modified L143R；And

L2 includes amino acid modified Q124E and V133E.

56. constructs according to claim 1, wherein

57. constructs according to claim 56, wherein

58. constructs according to claim 57, wherein

59. constructs according to claim 58, wherein：

H1 includes amino acid modified L143E, K145T, Q179E and S188L；

L1 includes amino acid modified Q124R and T178R；

H2 includes amino acid modified S186K；And

L2 includes amino acid modified Q124E, S176L and T180E.

60. constructs according to claim 58, wherein：

H1 includes amino acid modified L143E, K145T, Q179E and S188L；

L1 includes amino acid modified Q124R and T178R；

H2 includes amino acid modified S186K；And

L2 includes amino acid modified Q124E, S131T, T178Y and T180E.

61. constructs according to claim 58, wherein：

H1 includes amino acid modified L143E and K145T；

L1 includes amino acid modified Q124R, Q160K and T178R；

H2 includes amino acid modified S186K；And

L2 includes amino acid modified S131E.

62. constructs according to claim 58, wherein：

H1 includes amino acid modified L143E and K145T；

L1 includes amino acid modified Q124R；

H2 includes amino acid modified L143R；And

L2 includes amino acid modified Q124E and V133E.

63. constructs according to claim 1, wherein

64. constructs according to claim 63, wherein

65. constructs according to claim 64, wherein

H2 includes wild type or not modified amino acid sequence；And

L2 includes wild type or not modified amino acid sequence.

66. constructs according to claim 65, wherein：

H1 includes amino acid modified F122C and C233S；

L1 includes amino acid modified Q124C and C214S；

H2 includes wild type or not modified amino acid sequence；And

L2 includes wild type or not modified amino acid sequence.

75. according to construct in any one of the preceding claims wherein, wherein

79. constructs according to claim 77 or 78, wherein the Fc is heterodimer Fc.

B () reclaims the construct from the host cell cultures.

A () obtains H1, L1, H2 or L2；

C () obtains the construct.

E. cultivate at least one host cell to express the construct.

A kind of 115. computer-readable recording mediums, the media storage：

124. constructs according to claim 1, wherein the amino acid modified SMCA selected from table 35a designs 9561- 9095_1、9561-9095_2、9121-9373_1、9121-9373_2、9116-9349_1、9116-9349_2、9134-9521_ 1、9134-9521_2、9286-9402_1、9286-9402_2、9667-9830_1、9667-9830_2、9696-9848_1、 9696-9848_2、9060-9756_1、9060-9756_2、9682-9740_1、9682-9740_2、9049-9759_1、9049- 9759_2,9820-9823_1 and 9820-9823_2.

125. constructs according to claim 1, wherein the amino acid modified SMCA selected from table 35b designs 9327- 6054_1,9815-9825_1,9815-9825_2,9587-9735_1,9587-9735_2,3522_1,3522_2,3519_1 and 3519_2。